Fusogenic properties of saposin c and related proteins and peptides for application to transmembrane drug delivery systems

ABSTRACT

The present invention comprises a method for delivering pharmaceutical and/or imaging agents within and/or through the dermal, mucosal and other cellular membranes, and across the blood-brain barrier, utilizing a fusogenic protein. The fusogenic protein is associated with a phospholipid membrane, such as a liposome. The liposome may include dioleoylphosphatidylserine, a negatively charged long-chain lipid. Alternatively, the liposome is comprised of a mixture of negatively charged long-chain lipids, neutral long-chain lipids, and neutral short-chain lipids. Preferred fusogenic proteins include saposin C and other proteins, polypeptides and peptide analogs derived from saposin C. The active agent contained within the liposome may comprise biomolecules and/or organic molecules. This technology can be used for both cosmetic and medicinal applications in which the objective is delivery of the active agent within and/or beneath biological membranes or across the blood-brain barrier and neuronal membranes.

CROSS-REFERENCE TO RELATED APPLICATION

This application claims the benefit of U.S. Provisional PatentApplication Ser. No. 60/745,969 filed Apr. 28, 2006, which applicationis hereby incorporated by reference in its entirety.

This invention was made in part with Government support under Grant No.RO1 DK57690-01, awarded by the National Institutes of Health. TheGovernment may have certain rights in this invention.

FIELD OF INVENTION

The present invention relates to methods of delivering pharmaceutical ortherapeutic agents across biological membranes, where the agent iscontained within or intercalated into a phospholipid membrane anddelivery is facilitated by a membrane fusion protein. More particularly,the present invention relates to methods for enhancing the transport anddelivery of pharmaceutical agents across and/or within dermal andmucosal membranes or the blood-brain barrier, where the pharmaceuticalagent is contained within a liposome, and delivery is facilitated usingsaposin C, which is in association with the liposome.

BACKGROUND OF THE INVENTION

The therapeutic efficacy of pharmaceutical or therapeutic agents relieson the delivery of adequate doses of a pharmaceutical agent to the siteof action. Many modes of delivery have been developed, including, forexample, enteral (oral), parenteral (intramuscular, intravenous,subcutaneous), and topical administration. In most instances theadministration system is chosen for reliable dosage delivery andconvenience.

Typically, parenteral administration is the most reliable means ofdelivering a pharmaceutical to a patient. See, Goodman et al., Goodmanand Gilman's Pharmacological Basis of Therapeutics, Pergamon Press,Elmsford, N.Y. (1990) and Pratt et al. Principles of Drug Action: TheBasis of Pharmacology, Churchill Livingstone, New York, N.Y. (1990).Each parenteral mechanism insures that a prescribed dosage of thepharmaceutical agent is inserted into the fluid compartment of the bodywhere it can be transported. The disadvantage of these modes of deliveryis that they require an invasive procedure. The invasive nature ofadministration is inconvenient, painful and subject to infectiouscontamination.

Enteral and topical administration are more convenient, generallynon-painful, and do not predispose to infection, however both havelimited utility. The gastrointestinal and dermal surfaces presentformidable barriers to transport and therefore, some pharmaceuticalagents are not absorbed across these surfaces. Another drawback topatient directed modes of administration (enteral, topical andsubcutaneous) is compliance. Pharmaceutical agents that have a shorthalf-life require multiple daily doses. As the number of dosesincreases, patient compliance and therapeutic efficacy decrease.Simplified and/or less frequent administration schedules can aid inoptimizing patient compliance. Wilson et al. (1991) Harrison'sPrinciples of Internal Medicine, 12th Ed., McGraw-Hill, Inc., New York,N.Y.

The skin is an efficient barrier to the penetration of water solublesubstances, and the rate of transdermal pharmaceutical agent absorptionis primarily determined by the agent's lipid solubility, watersolubility, and polarity. Highly polar or water soluble pharmaceuticalagents are effectively blocked by the skin. Even very lipophilicpharmaceutical agents penetrate the dermis very slowly compared with therate of penetration across cell membranes. See Pratt et al. supra.

Efforts to develop more effective and convenient modes of pharmaceuticaladministration have led to the development of transdermal deliverysystems. Many current transdermal pharmaceutical agent delivery systemsrely upon pharmaceutical agents that are absorbed when admixed withinert carriers. See Cooper et al. (1987) “Penetration Enhancers”, inTransdermal Delivery of Drugs, Vol. II, Kyodonieus et al., Eds., CRCPress, Boca Raton, Fla. Few pharmaceutical agents fit this profile andthose which do are not always predictably absorbed. Various forms ofchemical enhancers, such as those enhancing lipophilicity, have beendeveloped to improve transdermal transport when physically mixed withcertain therapeutic agents and provide more predictable absorption. Seefor example, U.S. Pat. Nos. 4,645,502; 4,788,062; 4,816,258; 4,900,555;3,472,931; 4,006,218; and 5,053,227. Carriers have also been coupled topharmaceutical agents to enhance intracellular transport. See Ames etal. (1973) Proc. Natl. Acad. Sci. USA, 70:456-458 and (1988) Proc. Int.Symp. Cont. Rel. Bioact. Mater., 15:142.

Similar to the problems inherent in trans-dermal delivery ofpharmaceuticals, the blood-brain barrier is an obstacle to CNS drugdelivery. In fact, the blood-brain barrier is considered to be a“bottleneck” in brain drug development, and is perhaps the single mostimportant limitation on the future growth of neurotherapeutics.(Pardridge, W. M., The Blood-Brain Barrier: Bottleneck in Brain DrugDevelopment, The Journal of the American Society for ExperimentalNeuroTherapeutics, Vol 2, 3-14, January, 2005; Pardridge, W. M. Braindrug targeting: the future of brain drug development. Cambridge, UK:Cambridge University Press, 2001.) The BBB is formed by the braincapillary endothelium and prevents transport of approximately 100% oflarge-molecules (such as monoclonal antibodies, recombinant proteins,antisense or gene therapeutics) and more than 98% of all small-moleculedrugs into the brain. Although the average molecular mass of aCNS-active drug is 357 daltons, even a small, 100 dalton molecule suchas histamine does not pass through the BBB when infused into a mouse andallowed to distribute over thirty minutes time. In fact, a review of theComprehensive Medicinal Chemistry database shows that, of more than 7000small molecule drugs, only 5% treat the CNS, and this 5% treats onlydepression, schizophrenia, and insomnia.

Thus, most drugs do not cross the BBB. Unfortunately, many disorders ofthe central nervous system (CNS) could benefit from improved drugtherapy directed towards the CNS. While there is relatively littleresearch with respect to agents known to cross the BBB, there arecharacteristics that are predictive of a likelihood of success ofdelivery into the CNS. These are: 1) molecular mass under a 400-500Dalton threshold, and 2) high lipid solubility. Presently, only fourcategories of CNS disorders respond to such molecules, includingaffective disorders, chronic pain, and epilepsy. Migraine headache maybe considered a CNS disorder, and could also be included in thiscategory. In contrast, patients with diseases such as Alzheimer'sdisease, Parkinson's disease, Huntington's disease, A.L.S., multiplesclerosis, neuro-AIDS, brain cancer, stroke, brain or spinal cordtrauma, autism, lysosomal storage disorders, fragile X syndrome,inherited ataxias, and blindness have very limited options with respectto pharmaceutical treatments. (There has been some success with L-DOPAtreatment in Parkinson's patients, and multiple sclerosis can be treatedwith cytokines acting on the peripheral immune system.) (See generally,Partridge, supra).

In many of the above listed disorders, delivery across the BBB is therate limiting problem in gene therapy or enzyme replacement therapy.Many of these disorders could be treated with drugs, enzymes or genesalready discovered. However, these drugs do not cross the BBB and cannotbe considered for therapeutic use for that reason. Because of theimpermeability of the BBB, other approaches to drug delivery into theCNS must be used. These include the use of small molecules,trans-cranial brain drug delivery, and BBB disruption. However, none ofthese approaches provide solutions to the BBB problem that can bepractically implemented in a large number of patients. (Pardridge, W.M., “The Blood-Brain Barrier”)

Saposins, a family of small (˜80 amino acids) heat stable glycoproteins,are essential for the in vivo hydrolytic activity of several lysosomalenzymes in the catabolic pathway of glycosphingolipids (see Grabowski,G. A., Gatt, S., and Horowitz, M. (1990) Crit. Rev. Biochem. Mol. Biol.25, 385-414; Furst, W., and Sandhoff, K., (1992) Biochim. Biophys. Acta1126, 1-16; Kishimoto, Y., Kiraiwa, M., and O'Brien, J. S. (1992) J.Lipid. Res. 33, 1255-1267). Four members of the saposin family, A, B, C,and D, are proteolytically hydrolyzed from a single precursor protein,prosaposin (see Fujibayashi, S., Kao, F. T., Hones, C., Morse, H., Law,M., and Wenger, D. A. (1985) Am. J. Hum. Genet. 37, 741-748; O'Brien, J.S., Kretz, K. A., Dewji, N., Wenger, D. A., Esch, F., and Fluharty, A.L. (1988) Science 241, 1098-1101; Rorman, E. G., and Grabowski, G. A.(1989) Genomics 5, 486-492; Nakano, T., Sandhoff, K., Stumper, J.,Christomanou, H., and Suzuki, K. (1989) J. Biochem. (Tokyo) 105,152-154; Reiner, O., Dagan, O., and Horowitz, M. (1989) J. Mol.Neurosci. 1, 225-233). The complete amino acid sequences for saposins A,B, C and D have been reported as well as the genomic organization andcDNA sequence of prosaposin (see Fujibayashi, S., Kao, F. T., Jones, C.,Morse, H., Law, M., and Wenger, D. A. (1985) Am. J. Hum. Genet. 37,741-748; O'Brien, J. S., Kretz, K. A., Dewji, N., Wenger, D. A., Esch,F., and Fluharty, A. L. (1988) Science 241, 1098-1101; Rorman, E. G.,and Grabowski, G. A. (1989) Genomics 5, 486-492). A complete deficiencyof prosaposin with mutation in the initiation codon causes the storageof multiple glycosphingolipid substrates resembling a combined lysosomalhydrolase deficiency (see Schnabel, D., Schroder, M., Furst, W., Klien,A., Hurwitz, R., Zenk, T., Weber, J., Harzer, K., Paton, B. C., Poulos,A., Suzuki, K., and Sandhoff, K. (1992) J. Biol. Chem. 267, 3312-3315).

Saposins are defined as sphingolipid activator proteins or coenzymes.Structurally, saposins A, B, C, and D have approximately 50-60%similarity including six strictly conserved cysteine residues (seeFurst, W., and Sandhoff, K., (1992) Biochim. Biophys. Acta 1126, 1-16)that form three intradomain disulfide bridges whose placements areidentical (see Vaccaro, A. M., Salvioli, R., Barca, A., Tatti, M.,Ciaffoni, F., Maras, B., Siciliano, R., Zappacosta, F., Amoresano, A.,and Pucci, P. (1995) J. Biol. Chem. 270, 9953-9960). All saposinscontain one glycosylation site with conserved placement in theN-terminal sequence half, but glycosylation is not essential to theiractivities (see Qi. X., and Grabowski, G. A. (1998) Biochemistry 37,11544-11554; Vaccaro, A. M., Ciaffoni, F., Tatti, M., Salvioli, R.,Barca, A., Tognozzi, D., and Scerch, C. (1995) J. Biol. Chem. 270,30576-30580). In addition, saposin A has a second glycosylation site inC-terminal half.

All saposins and saposin-like proteins and domains contain a “saposinfold” when in solution. This fold is a multiple α-helical bundle motif,characterized by a three conserved disulfide structure and severalamphipathic polypeptides. Despite this shared saposin-fold structure insolution, saposins and saposin-like proteins have diverse in vivobiological functions in the enhancement of lysosomal sphingolipid (SL)and glycosphingolipid (GSL) degradation by specific hydrolases. Becauseof these roles, the saposins occupy a central position in the control oflysosomal sphingolipid and glycosphingolipid metabolisms (see Kishimoto,Y., Kiraiwa, M., and O'Brien, J. S. (1992) J. Lipid. Res. 33, 1255-1267;Fujibayashi, S., Kao, F. T., Hones, C., Morse, H., Law, M., and Wenger,D. A. (1985) Am. J. Hum. Genet. 37, 741-748; O'Brien, J. S., Kretz, K.A., Dewji, N., Wenger, D. A., Esch, F., and Fluharty, A. L. (1988)Science 241, 1098-1101).

The structural characteristic of these saposins is of great importanceto the diverse mechanisms of activation. Since all of these proteinshave high sequence similarity, but different mechanisms of action withlipid membranes, one can speculate that the specific biologicalfunctions of saposins and saposin-like proteins are the result of thedifferential interactions with the biological membrane environments. Invitro, saposin A enhances acid β-glucosidase activity at μMconcentration, but saposin C deficiency leads to glucosylceramidestorage and a “Gaucher disease-like” phenotype (see Schnable, D.,Schroder, M., and Sandhoff, K. (1991) FEBS Lett. 284, 57-59; Rafi. M.A., deGala, G., Zhang, X. L., and Wenger, D. A. (1993) Somat. Cell Mol.Genet. 19, 1-7). Activation of saposin B takes place throughsolubilizing and presenting glycosphingolipid substrates to lysosomalenzymes (see Furst, W., and Sandhoff, K., (1992) Biochim. Biophys. Acta1126, 1-16).

Saposin C promotes acid β-glucosidase activity by inducing in the enzymeconformational change at acidic pH (see Berent, S. L., and Radin, N. S.(1981) Biochim. Biophys. Acta 664, 572-582; Greenberg, P., Merrill, A.H., Liotta, D. C., and Grabowski, G. A. (1990) Biochim. Biophys. Acta1039, 12-20; Qi. X., and Grabowski, G. A. (1998) Biochemistry 37,11544-11554). This interaction of saposin C with the enzyme occurs onnegatively charged phospholipid surfaces. In vitro and ex vivo saposinsA and D function to enhance the degradation of galactosylceramide andceramide/sphingomyelin, respectively (see Harzer, K., Paton, B. C.,Christomanou, H., Chatelut, M., Levade, T., Hiraiwa, M. and O'Brien, J.S. (1997) FEBS Lett. 417, 270-274; Klien, A., Henseler, M., Klein, C.,Suzuki, K., Harzer, K., and Sandhoff, K. (1994) Biochem. Biophys. Res.Commun, 200, 1440-1448). Patients lacking the individual saposins B andC showed a variant form of metachromatic leukodystrophy and Gaucherdisease, respectively. (see Wenger, D. A., DeGala, G., Williams, C.,Taylor, H. A., Stevenson, R. E., Pruitt, J. R., Miller, J., Garen, P.D., and Balentine, J. D. (1989) Am. J. Med. Genet. 33, 255-265) (seeChristomanou, H., Aignesberger, A., and Linke, R. P. (1986) Biol. Chem.Hoppe-Seyler 367, 879-890).

The primary physiological function of saposin C has been defined by aglycosphingolipid (GSL) storage disease similar to neuronopathic“Gaucher's disease” in patients with a deficiency of the protein.Saposin C is a critical physiologic activator for the lysosomal enzyme,acid β-glucosidase. In addition to stimulating the glucosylceramidedegradation by acid β-glucosidase, saposin C has several other potentialroles. These include inter-membrane transport of gangliosides and GSLs,reorganization and destabilization of phospholipids-containingmembranes, and fusion of acid phospholipids vesicles (see Hiraiwa, M.,and Soeda, S. et al. (1992) Proc. Natl. Acad. Sci. USA, 89, 11254-11258;You, H. X., and Yu, L. et al., (2001) FEBS Left. 503, 97-102; You, H. X.and Qi, X. et al. (2003) Biophys. J. 84, 2043-2057; Vaccaro, A. M., andTatti, M. et al., (1994) FEBS Lett. 349, 181-186; Wang, Y., andGrabowski, G. et al., Biochem. Biophys., 415: 43-53; Qi, X. and Chu, Z.,(2004) Arch. Biochem. Biophys., 424: 210-218). Saposin C associates withphophatidyserine (PS) membranes by embedding its amino- and carboxyl-endhelices into the outer leaflet of membranes (see Qi, X and Grabowski, G.A., (2001) J. Biol. Chem., 276, 27010-27017). Increasing evidenceindicates that intereactions of saposins with appropriate membranes arecrucial for their specificity and activity.

Moreover, PSAP, the precursor of saposins, is a neurotropic factor within vitro neuritogenic, in vivo nerve growth promoting, and apoptosisprotection properties (see Qi, X. and Qin, W. et al. (1996) J. Biol.Chem, 217, 6874-6880; O'Brien, J. S. and Carson, G. S. et al. (1994)Proc. Natl. Acad. Sci. USA 91, 9593-9596; Qi, X. and Kondoh, K. et al.(1999) Biochemistry 38, 6284-6291; Kotani, Y. S. and Matsuda, S. et al.(1996) J. Neurochem. 66, 2019-2025; Koani, Y. and Matsuda, S. et al.(1996) J. Neurochem. 66, 2197-2200; Tsuboi, K. and Hiraiwa, M. et al.(1998) Brain Res. Dev. Brain Res. 110, 249-255). Such neuritogenicfunctions are mediated through sequences in the NH₂-terminal half ofsaposin C (see Qi, X. and Qin, W. et al. (1996) J. Biol. Chem. 271,6874-6880; O'Brien, J. S. and Carson, G. S. et al. (1995) FASEB J. 9,681-685). The minimum sequence required for in vitro neuritogenicactivity spans amino acid residues 22-31 of saposin C in humans andmice. Neurological functions of PSAP and saposin C are mediated byactivation of the enzymes in the MAPK pathway through aG-protein-associated cell membrane receptor in a number ofneuroglia-derived cells (see Campana, W. M. and Hiraiwa, M. et al.(1996) Biochem. Biophys. Res. Commun. 229, 706-712; Hiraiwa, M. andCampana, W. M. et al. (1997) Biochem. Biophys. Res. Commun. 240,415-418).

Human and mouse PSAP genetic defects result in total saposin deficiency(see Harzer, K. and Paton, B. C. et al. (1989) Eur. J. Pediatr. 149,31-39; Hulkova, H., and Cervenkova, M. et al. (2001) Hum. Mol. Genet.10, 927-940; Fujita, N. and Suzuki, K. et al., Hum. Mol. Genet. 5,711-725). This deficiency can lead to aberrant accumulation ofmultivesicular bodies (MVBs), as observed in the skin fibroblasts fromPSAP-deficient patients (see Harzer, K. and Paton, B. C. et al. (1989)Eur. J. Pediatri. 149, 31-39; Burkhardt, J. K. and Huttler, S. et al.(1997) Eur. J. Cell Biol. 73, 10-18). Further, the sinusoidal cells inliver from a PSAP-deficient patient has been observed to be crowded withmultivesicular inclusions (see Sandhoff, K. and Kolter, T. et al. (2000)The Metabolic and Molecular Bases of Inherited Disease, 3371-3388;Harzer, K. and Paton, B. C. et al. (1989) Eur. J. Pediatr. 149, 31-39).Similar MVB structures also were found in fibroblasts from a saposinC-deficient patient (see Pampols, T. and Pineda, M. et al. (1999) ActaNeuropathol. 97, 91-97). In PSAP−/− (double-knock out) mice, inclusionsconsisting of numerous concentric lamellar bodies and dense granularstructures were noted in a variety of tissues and cells (see Oya, Y.,and Nakayasu, H. et al. (1998) Acta Neuropathol 96, 29-40). Thinsections of mouse PSAP−/− cells revealed a selective accumulation ofMVBs by electron microscopy (see Morales, C. R. and Zhao, Q. et al.(1999) Biocell 23, 149-160).

MVBs, a subset of the late endosomes, have a crucial role incommunications by vesicular transport between the trans-Golgi network,the plasma membrane, and lysosomal/vacuolar organelles (see Katzman, D.J. and Odorizzi, G. et al. (2002) Nat. Rev. Mol. Cell Biol. 3, 893-905).One function of MVBs is to maintain the cellular homeostasis requiredfor neuronal development and growth. The hypothetical “signalingendosome” model explains that the ligand-receptor complex on anendosomal signaling platform is transported retrogradely from the distalaxon to the cell body to promote gene expression and neuron survival(see Ginty, D. D. and Segal, R. A. (2002) Curr. Opin. Neurobiol. 12,268-274). The abnormalities in MVB structures in neurons of PSAP−/− micemay disrupt the retrograde movement of neurotrophins via vesicularsignaling transports and may impair the development of neuronal cells inthe CNS.

Introducing exogenous PSAP or saposin C into the medium of culturedfibroblasts from the PSAP-deficient patient reverses the aberrantaccumulation of MVBs, suggesting that saposin C is a key regulatorymolecule in MVB formation (see Burkhardt, J. K. and Huttler, S. et al.(1997) Eur. J. Cell Biol. 73 10-18; Chu, Z., and Witte, D. P. et al.(2004) Exp. Cell Res.).

In addition to mediating MVB formation, saposin plays a role in membranefusion. Membrane fusion is a major event in biological systems drivingsecretion, endocytosis, exocytosis, intracellular transport,fertilization, and muscle development (see Christomanou, H., Chabas, A.,Pampols, T., and Guardiola, A. (1989) Klin, Wochenschr. 67, 999-1003).Recent experimental evidence generated by this inventor has indicatedthat saposin-lipid membrane interactions play a critical role insaposin-mediated membrane fusion of lipids thereby facilitatingtransport of active agents across these biological membranes.

The present invention also relates to a method of administering imagingagents across cellular membranes including the blood-brain barrier usingsaposin C containing liposomes. Non-invasive imaging techniques can beused to monitor the distribution and efficacy of liposomal deliverysystems, thereby facilitating the evaluation and clinical application ofgene therapy or therapeutic treatment using liposomes. Imaging agentsmay use magnetic resonance, fluorescence, or CT/PET as a means ofdetection. However, key obstacles to successful use of imaging agents tomonitor liposome delivery are ease of detection, availability ofpertinent technology and ease and efficiency of delivery.

With respect to using liposomes to deliver imaging agents, lipophilicmolecules are generally appropriate, though the present invention is notlimited to use with such molecules. Without intending to be limited bytheory, lipophilic dyes or dyes containing a lipophilic moiety mayintercalate into the liposomal membrane or reconstitute into the lipidcore of liposomal structures. Examples of such dyes known in the art arethe indocarbocyanine dye, DiI. DiI is a fluorescent carbocyanine dyethat is routinely used to label lipid membranes. Other similar dyes areDiA or DiaO as described in Honig, M. G. et al, DiI and DiO: versatilefluorescent dyes for neuronal labeling and pathway tracing. TrendsNeurosci. 12:333-335, 340-331, 1989. Other lipophilic dyes that may beused with the present invention include PKH2, NeuroVue Green, PKH 26,NeuroVue Red, and NeuroVue Maroon, as described by Fritzsch, et al.Diffusion and Imaging proerties of Three New Lipophilic Tracers,NeuroVue Maroon, NeuroVue and Neurovue Green and their use for Doubleand Triple Labeling of Neuronal Profile, manuscript. Any of these dyesmay be used with the present invention described herein, either alone orin combination.

Also used in the art and appropriate to the present invention areimaging agents having two or more imaging properties. Such agents allowthe researcher or clinician the ability to use multiple methods ofimaging to detect administered imaging agents. An example of such agentsare the so-called PTIR dyes as described by Li, H., et al., MR andFluorescent Imaging of Low-Density Lipoprotein Receptors, Acad Radiol.2004; 11:1251-1259, incorporated herein by reference. These dyes containboth a fluorophore and a Gd(III) moiety that allow for detection viamagnetic resonance imaging (MRI) or confocal fluorescence microsopy. Thelipophilic side chain facilitates the intercalation of the dye intophospholipid monolayers. Thus, these dyes are appropriate for use withliposomal delivery systems such as the one described herein.

Proton MR imaging offers the advantages of being noninvasive,tomographic, and of high resolution. In recent years, magnetic resonanceimaging (MRI) has emerged as a powerful tool in clinical settingsbecause it is noninvasive and yields an accurate volume rendering of thesubject. See generally, U.S. Pat. No. 6,962,686 Kayyem, et al. entitledCell-specific gene delivery vehicles. These advantages make MRI thetechnique of choice in both medical imaging and as an imaging tool foruse in biological experiments. Unlike light-microscope imagingtechniques based upon the use of dyes or fluorochromes, MRI does notproduce toxic photobleaching by-products. Furthermore, unlikelight-microscopy, MRI is not limited by light scattering or otheroptical aberrations to cells within approximately only one hundredmicrons of the surface. Agents having MRI properties such as thosedescribed above may be used with the present invention.

Accordingly, there exists a significant need for nontoxic agents whichcan improve the delivery or transport of pharmaceutical or imagingagents across or through biological membranes, including the blood-brainbarrier. The present invention fulfills these needs.

SUMMARY OF THE INVENTION

The present invention relates to methods of delivering agents, e.g.,pharmaceutical or therapeutic agents, across biological membranes, wherethe agent is contained within or intercalated into a phospholipidmembrane and delivery is facilitated by a membrane fusion protein. Moreparticularly, the present invention relates to methods for enhancing thetransport and delivery of agents across and/or within dermal and mucosalmembranes or the blood-brain bather, where the agent is contained withina liposome, and delivery is facilitated using saposin C, which is inassociation with the liposome.

As described herein, the present invention comprises a method fordelivering a pharmaceutical agent through a biological membrane,including the blood-brain barrier and cellular membranes, wherein themethod comprises applying to the membrane a composition comprisinganionic phospholipids with or without neutral phospholipids, a safe andeffective amount of the pharmaceutical agent contained within thephospholipids, and a fusogenic protein or polypeptide derived fromprosaposin in a pharmaceutically acceptable carrier.

In one embodiment, the anionic phospholipid membrane is a vesicle. Inanother embodiment, the vesicle is a liposome. The liposomes are a formof nanocontainer and nanocontainers, such as nanoparticles or liposomes,are commonly used for encapsulation of drugs. Cationic phospholipids mayalso be used, provided that the overall charge of the resulting liposomeis negative.

In another embodiment of the present invention, the pH of theprotein-lipid composition is acidic. In another embodiment of thepresent invention, the pH of the composition is between about 6.8 and 2.In another embodiment of the present invention, the pH of thecomposition is between about 5.5 and 2. In another embodiment, the pH isbetween about 5.5 and about 3.5.

In another embodiment, the protein and lipid composition is provided ina dry form, e.g., a powder. In another embodiment, the protein and lipidcomposition dry form is treated with an acid. In one embodiment, theacid is an acidic buffer or organic acid. In another embodiment, theacid is added at a level sufficient to protonate at least a portion ofthe protein, wherein the composition has a pH of from about 5.5 andabout 2. In another embodiment, the acid is added at a level sufficientto substantially protonate the protein, wherein the composition has a pHof from about 5.5 and about 2.

In a further embodiment, the pH of the protein and lipid composition drypowder that has been treated with an acid sufficient to protonate atleast a portion of the protein is then substantially neutralized. In oneembodiment, the pH is neutralized with a neutral pH buffer. In oneembodiment, the pH is neutralized with a neutral pH buffer sufficientlyto control the size of the resulting liposome. In another embodiment,the pH is neutralized with a neutral pH buffer sufficiently to controlthe size of the resulting liposome to provide for liposomes having meandiameters of about 200 nanometers. In another embodiment, the liposomeshave a mean diameter of between 50 and 350 nanometers. In anotherembodiment, the liposomes have a mean diameter of between 150 and 250nanometers. In another embodiment, the buffer is added to thecomposition to provide a final composition pH of from about 5 to about14, preferably from about 7 to 14, more preferably from about 7 to about12, more preferably from about 7 to about 10, and even more preferablyfrom about 8 to about 10.

In one embodiment of the present invention, short-chain lipids are used.Generally, the concentration of the fusogenic protein or polypeptide isof a sufficient amount to deliver the pharmaceutical agent within and/orthrough the membrane. In another embodiment, the concentration ofphospholipids in in vitro membranes is in at least a 5-fold excess tothat of the fusogenic protein or polypeptide by molar ratio. In anotherembodiment, the concentration of phospholipids in in vitro membranes isin at least a 10-fold excess to that of the fusogenic protein orpolypeptide by molar ratio. In another embodiment, the concentration ofphospholipids in in vitro membranes is in at least a 15-fold excess tothat of the fusogenic protein or polypeptide by molar ratio. In oneembodiment, the concentration of phospholipids in in vitro membranes isin at least a 20-fold excess to that of the fusogenic protein orpolypeptide by molar ratio. In another embodiment, the concentration ofphospholipids in in vitro membranes are in about a 10 to about 50-foldexcess or about 20 to about 30 fold excess to that of the fusogenicprotein or polypeptide by molar ratio.

In one embodiment, the concentration of phospholipids in in vivo or cellmembrane systems are in at least a 1-fold excess to that of thefusogenic peptide by molar ratio. In one embodiment, the concentrationof phospholipids in in vivo or cell membrane systems are in at least a2-fold excess to that of the fusogenic peptide by molar ratio. Inanother embodiment, the concentration of phospholipids in in vivo orcell membrane systems are in at least a 3-fold excess to that of thefusogenic peptide by molar ratio. In another embodiment, theconcentration of phospholipids in in vivo or cell membrane systems arein about a 1 to about a 10 fold excess or about 3 to 7 fold excess tothat of the fusogenic peptide by molar ratio.

Without wishing to be bound by theory in any way, it is believed thatthe membrane fusion protein is associated with the phospholipidmembrane, through electrostatic and hydrophobic and hydrophobicinteractions and the overall charge of the lipid composition isnegative.

In accordance with the present invention, the targeted biologicalmembranes include, but are not limited to, dermal membranes, mucosalmembranes, the blood-brain barrier and cellular membranes.

The preferred membrane fusion proteins include saposin C as well asother proteins, polypeptide analogues or polypeptides derived fromeither saposin C, SEQ. ID. NO. 1 through 13 and mixtures thereof.

In one embodiment, the membrane fusion protein comprises at least 8, 10,12, 14, 16, 18, 20, 22, 24 or more contiguous amino acids of a sequenceselected from saposin C, SEQ. ID. NO. 1 and SEQ. ID. NO. 2. In oneembodiment, the membrane fusion protein comprises a peptide of theformula:

h-u-Cys-Glu-h-Cys-Glu-h-h-h-Lys-Glu-h-u-Lys-h-h-Asp-Asn-Asn-Lys-u-Glu-Lys-Glu-h-h-Asp-h-h-Asp- Lys-h-Cys-u-Lys-h-h

-   -   where h=hydrophobic amino acids, including, Val, Leu, Ile, Met,        Pro, Phe, and Ala; and where u=uncharged polar amino acids,        including, Thr, Ser, Tyr, Gly, Gln, and Asn, and mixtures        thereof.

In another embodiment, the membrane fusion protein comprises one or moreprotein selected from other proteins, polypeptide analogues orpolypeptides derived from either saposin C, SEQ. ID. NO. 1, SEQ. ID. NO.2, polypeptides of the formula may be used:

h-u-Cys-Glu-h-Cys-Glu-h-h-h-Lys-Glu-h-u-Lys-h-h-Asp-Asn-Asn-Lys-u-Glu-Lys-Glu-h-h-Asp-h-h-Asp- Lys-h-Cys-u-Lys-h-h,

-   -   where h=hydrophobic amino acids, including, Val, Leu, Ile, Met,        Pro, Phe, and Ala; and where u=uncharged polar amino acids,        including, Thr, Ser, Tyr, Gly, Gln, and Asn, and mixtures        thereof.

In one embodiment, the membrane fusion protein comprises at least 8, 10,12, 14, 16, 18, 20, 22, 24, 30 or more contiguous amino acids of thesequence: Ser Asp Val Tyr Cys Glu Val Cys Glu Phe Leu Val Lys Glu ValThr Lys Leu Ile Asp Asn Asn Lys Thr Glu Lys Glu Ile Leu Asp Ala Phe AspLys Met Cys Ser Lys Leu Pro. the membrane fusion protein comprises atleast 8, 10, 12, 14, 16, 18, 20, 22, 24, 30 or more contiguous aminoacids of the sequence: Val Tyr Cys Glu Val Cys Glu Phe Leu Val Lys GluVal Thr Lys Leu Ile Asp Asn Asn Lys Thr Glu Lys Glu Ile Leu Asp Ala PheAsp Lys Met Cys Ser Lys Leu Pro.

In another embodiment, the membrane fusion protein comprising thesequence: Ser Asp Val Tyr Cys Glu Val Cys Glu Phe Leu Val Lys Glu ValThr Lys Leu Ile Asp Asn Asn Lys Thr Glu Lys Glu Ile Leu Asp Ala Phe AspLys Met Cys Ser Lys Leu Pro. In another embodiment, the membrane fusionprotein comprising the sequence: Val Tyr Cys Glu Val Cys Glu Phe Leu ValLys Glu Val Thr Lys Leu Ile Asp Asn Asn Lys Thr Glu Lys Glu Ile Leu AspAla Phe Asp Lys Met Cys Ser Lys Leu Pro. In another embodiment, themembrane fusion protein is a 22-mer comprising the sequence:CEFLVKEVTKLIDNNKTEKEIL.

In another embodiment, the membrane fusion protein comprises saposin C.In another embodiment, the membrane fusion protein comprises saposin Cin an oxidized, acetylated (for example, formylated and acetylated),acetoacetylated or lactosylated form by modification according to knownmethods (see, in particular, J. M. Shaw, op. cit.; Basu et al., Proc.Natl. Acad. Sci. USA 73, 3178-3182 (1976); J. Steinbrechert, Biol. Chem.262, 3703 (1987)).

In one embodiment, the phospholipid membranes are anionic liposomescontaining the pharmaceutical agent. In another embodiment, theliposomes are made from any mixture of lipids that contain anioniclong-chain lipids. In another embodiment, the liposomes are made from amixture containing anionic long-chain lipids, neutral long-chain lipids,and neutral short-chain lipids, wherein the overall charge of theresulting liposome is negative.

In selecting a lipid for preparing the liposomes used in the presentinvention, a wide variety of lipids will be found to be suitable fortheir construction. Particularly useful are any of the materials orcombinations thereof known to those skilled in the art as suitable forliposome preparation. The lipids used may be of natural, synthetic orsemi-synthetic origin.

In another embodiment, the liposomes are made from one or morebiocompatible lipid. In another embodiment, the biocompatible lipid isselected from the group consisting of fatty acids, lysolipids,phosphatidylcholines, phosphatidylethanolamines, phosphatidylserines,phosphatidylglycerols, phosphatidylinositols, sphingolipids,glycolipids, glucolipids, sulfatides, glycosphingolipids, phosphatidicacids, palmitic acids, stearic acids, arachidonic acids, oleic acids,lipids bearing polymers, lipids bearing sulfonated monosaccharides,lipids bearing sulfonated disaccharides, lipids bearing sulfonatedoligosaccharides, lipids bearing sulfonated polysaccharides,cholesterols, tocopherols, lipids with ether-linked fatty acids, lipidswith ester-linked fatty acids, polymerized lipids, diacetyl phosphates,dicetyl phosphates, stearylamines, cardiolipin, phospholipids with fattyacids of 6-8 carbons in length, synthetic phospholipids with asymmetricacyl chains, ceramides, non-ionic lipids, sterol aliphatic acid esters,sterol esters of sugar acids, esters of sugar acids, esters of sugaralcohols, esters of sugars, esters of aliphatic acids, saponins,glycerol dilaurate, glycerol trilaurate, glycerol dipalmitate, glycerol,glycerol esters, alcohols of 10-30 carbons in length,6-(5-cholesten-3beta-yloxy)-1-thio-beta-D-galactopyranoside,digalactosyldiglyceride,6-(5-cholesten-3beta-yloxy)hexyl-6-amino-6-deoxy-1-thio-beta-D-galactopyranoside,6-(5-cholesten-3beta-yloxy)hexyl-6-amino-6-deoxyl-1-thio-alpha-D-mannopyranoside,12-(((7′-diethylaminocoumarin-3-yl)carbonyl)methylamino)-octadecanoicacid,N-[12-(((7′-diethylaminocoumarin-3-yl)carbonyl)methyl-amino)octadecanoyl]-2-aminopalimiticacid, cholesteryl(4′-trimethyl-ammonio)butanoate,N-succinyldioleoylphosphatidylethanol-amine, 1,2-dioleoyl-sn-glycerol,1,2-dipalmitoyl-sn-3-succinylglycerol,1,3-dipalmitoyl-2-succinylglycerol,1-hexadecyl-2-palmitoylglycerophosphoethanolamine,palmitoylhomocysteine, cationic lipids,N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammoium chloride,1,2-dioleoyloxy-3-(trimethylammonio)propane,1,2-dioleoyl-3-(4′-trimethyl-ammonio)butanoyl-sn-glycerol,lysophospholipids, lysobisphosphatidic acid (LBPA),semi-lysobisphosphatidic acid (semi-LBPA), cardiolipin, lipids bearingcationic polymers, alkyl phosphonates, alkyl phosphinates, and alkylphosphites.

In one embodiment, the phosphatidylcholine is selected from the groupconsisting of dioleoylphosphatidylcholine,dimyristoylphosphatidylcholine, dipentadecanoylphosphatidylcholine,dilauroylphosphatidylcholine, dipalmitoylphosphatidylcholine, anddistearoylphosphatidylcholine; wherein the phosphatidylethanolamine isselected from the group consisting ofdipalmitoylphosphatidylethanolamine anddioleoylphosphatidylethanolamine; wherein the sphingolipid issphingomyelin; wherein the glycolipid is selected from the groupconsisting of ganglioside GM1 and ganglioside GM2; wherein in the lipidsbearing polymers the polymer is selected from the group consisting ofpolyethyleneglycol, chitin, hyaluronic acid and polyvinylpyrrolidone;wherein the sterol aliphatic acid esters are selected from the groupconsisting of cholesterol sulfate, cholesterol butyrate, cholesterolisobutyrate, cholesterol palmitate, cholesterol stearate, lanosterolacetate, ergosterol palmitate, and phytosterol n-butyrate; wherein thesterol esters of sugar acids are selected from the group consisting ofcholesterol glucuronide, lanosterol glucuronide, 7-dehydrocholesterolglucuronide, ergosterol glucuronide, cholesterol gluconate, lanosterolgluconate, and ergosterol gluconate; wherein the esters of sugar acidsand the esters of sugar alcohols are selected from the group consistingof lauryl glucuronide, stearoyl glucuronide, myristoyl glucuronide,lauryl gluconate, myristoyl gluconate, and stearoyl gluconate; whereinthe esters of sugars and the esters of aliphatic acids are selected fromthe group consisting of sucrose laurate, fructose laurate, sucrosepalmitate, sucrose stearate, glucuronic acid, gluconic acid, accharicacid, and polyuronic acid; wherein the saponins are selected from thegroup consisting of sarsasapogenin, smilagenin, hederagenin, oleanolicacid, and digitoxigenin; wherein the glycerol esters are selected fromthe group consisting of glycerol tripalmitate, glycerol distearate,glycerol tristearate, glycerol dimyristate, glycerol and trimyristate;wherein the alcohols are of 10-30 carbon length and are selected fromthe group consisting of n-decyl alcohol, lauryl alcohol, myristylalcohol, cetyl alcohol, and n-octadecyl alcohol; wherein in the lipidsbearing cationic polymers the cationic polymers are selected from thegroup consisting of polylysine and polyarginine.

In another embodiment, the lipid is selected from the group consistingof dipalmitoylphosphatidylcholine, dipalmitoylphosphatidylethanolamine,and dipalmitoylphosphatidic acid.

In another embodiment, the pharmaceutical agent contained within theliposome comprises biomolecules and/or organic molecules. Thistechnology can be used for both cosmetic and medicinal applications inwhich the objective is delivery of the active agent across membrane suchas the dermal, mucosal, blood-brain barrier, or cellular membranes.

The present invention also relates to a method of treating disease bytransporting macromolecules such as genes, proteins, and otherbiological or organic molecules across the blood-brain barrier whereinthe method comprises the administration of a composition comprisinganionic liposomes with or without short-chain lipids, a safe andeffective amount of a macromolecular therapeutic agent contained withinthe liposomes and saposin C.

In further embodiments, the instant invention features compositionscomprising a small nucleic acid molecule, such as short interferingnucleic acid (siNA), a short interfering RNA (siRNA), a double-strandedRNA (dsRNA), micro-RNA (mRNA), or a short hairpin RNA (shRNA), admixedor complexed with, or conjugated to, a saposin fusogenic membrane orliposome.

The present invention also relates to a method by which neuroblastoma,cerebral inflammation, metachromatic leukodystrophy (MLD), Niemann-Pick,stroke, Parkinson's, Alzheimer's diseases, demyelination disorders,retinal neuropathy, Huntington's disease, A.L.S., multiple sclerosis,neuro-AIDS, brain cancer, brain or spinal cord trauma, autism, lysosomalstorage disorders, fragile X syndrome, inherited ataxias, and blindnesscan be treated in which the method comprises the steps of making aliposomal delivery system in which the liposome is comprised of acidiclong-chain lipids, with or without the addition of neutral long-chainlipids and neutral short-chain lipids, and saposin C, prosaposin, aswell as other proteins, polypeptide analogues or polypeptides derivedfrom saposin C or prosaposin. The liposome can contain therapeuticagents such as anti-inflammatory agents, anti-apoptotic, andneuroprotective agents, or enzymes, proteins, or the correspondinggenes, DNA or RNA sequences for genes identified as lacking in thesediseases.

In one embodiment, compositions and methods are provided comprising apolynucleotide, or a precursor thereof, in combination with fusogenicanionic phospholipid membranes or liposomes, a fusogenic protein orpolypeptide derived from prosaposin, and a pharmaceutically acceptablecarrier.

In one embodiment, compositions and methods are provided comprising ashort interfering nucleic acid (siNA), or a precursor thereof, incombination with fusogenic anionic phospholipid membranes or liposomes,a fusogenic protein or polypeptide derived from prosaposin, and apharmaceutically acceptable carrier. Within the novel compositions ofthe invention, the siNA may be admixed or complexed with, or conjugatedto, the fusogenic anionic phospholipid membranes or liposomes with afusogenic protein or polypeptide derived from prosaposin to form acomposition that enhances intracellular delivery of the siNA.

The present invention also comprises a method for treating Gaucher'sDisease, wherein the method comprises the administration of acomposition comprising anionic liposomes, a safe and effective amount ofacid beta-glucosidase contained within the liposomes; and saposin C, allcontained in a pharmaceutically acceptable carrier, wherein the pH ofthe composition is about 7, 6.8, 6.5, 6, 5.9, 5.8, 5.7, 5.6, 5.5, 5.4,5.3, 5.2, 5.1, 5.0 or less and the saposin C is associated with thesurface of the liposome through an electrostatic and hydrophobicinteraction. Generally, the concentration of the liposome is about a 1to 10-fold excess to that of saposin C. In one embodiment, the pH of thecomposition is less than about 6.8. In another embodiment, the pH of thecomposition is less than about 6.0. In another embodiment, the pH of thecomposition is less than about 5.5. In another embodiment, the pH of thecomposition is less than about 5.0.

The present invention also relates to a method for treating Peyer'spatches, mesenteric lymph nodes, bronchial lymph nodes wherein themethod comprises the administration of a composition comprising anioniclong-chain lipids, long-chain neutral lipids and/or neutral short-chainlipids, a safe and effective amount of lipid, DNA or protein antigens,saposin C, prosaposin, as well as other proteins, polypeptide analoguesor polypeptides derived from saposin C or prosaposin.

The present invention also relates to a method of imaging tissues andcells wherein the composition is comprised of a saposin-C containingliposome and a detectable imaging agent selected from the groupconsisting of MRI detectable agents, fluorescent agents, CT/PETdetectable agents, agents having multiple detection properties, orcombinations thereof. The agent can be either intercalated into thelipid membrane or encapsulated within the liposome. In anotherembodiment of the present invention, the saposin C liposomal complex canincorporate one, two or three distinct agents having different imagingproperties such that multiple, distinct detection methods can be usedwith a single administration of saposin C liposomes.

Other ancillary agents include fluorophores (such as fluorescein,dansyl, quantum dots, and the like) and infrared dyes or metals may beused in optical or light imaging (e.g., confocal microscopy andfluorescence imaging).

In another embodiment, the composition further comprises a radionuclide,a chelating agent, biotin, a fluorophore, an antibody, horseradishperoxidase, alkaline phosphatase, nanoparticles, quantum dots,nanodroplets of anticancer agents, anticancer agents or chemotherapeuticagents, liposomal drugs, cytokines or small molecule toxins attachedthereto.

In another embodiment, the imaging moiety is selected from the groupconsisting of a radionuclide, biotin, a fluorophore, an antibody,horseradish peroxidase, alkaline phosphatase, nanoparticles, quantumdots, nanodroplets of detectable anticancer agents, liposomal drugs andcytokines.

One of ordinary skill is familiar with methods for attachingradionuclides, chelating agents, and chelating agent-linker conjugatesto the ligands of the present invention. In particular, attachment ofradionuclides, chelating agents, and chelating agent-linker conjugatesto the ligands of the present invention can be conveniently accomplishedusing, for example, commercially available bifunctional linking groups(generally heterobifunctional linking groups) that can be attached to afunctional group present in a non-interfering position on the compoundand then further linked to, for example, a radionuclide,chemotherapeutic agent, anticancer agent, nanoparticle, quantum dot,nanodroplet of an anticancer agent or a small molecule toxin. In thismanner, the compounds of the present invention can be used to carrysuitable agents to a target site, generally, a tumor or organ or tissuehaving cancerous cells. In another embodiment, a ligand Qdot complex Isprepared by incubating biotinylated ligand with streptavidin-Qdot605(Quantum Dot Corp.; Hayward, Calif.).

In one embodiment of this invention, the liposome containing a traceableimaging agent can be used to target tumors such as neuroblastoma,allowing for determination of tumor size, growth, location ormetastasis.

The above summary of the present invention is not intended to describeeach embodiment or every implementation of the present invention.Advantages and attainments, together with a more complete understandingof the invention, will become apparent and appreciated by referring tothe following detailed description and claims taken in conjunction withthe accompanying drawings.

Throughout this document, all temperatures are given in degrees Celsius,and all percentages are weight percentages unless otherwise stated. Allpublications mentioned herein are incorporated herein by reference forthe purpose of describing and disclosing the compositions andmethodologies which are described in the publications which might beused in connection with the presently described invention. Thepublications discussed herein are provided solely for their disclosureprior to the filing date of the present application. Nothing herein isto be construed as an admission that the invention is not entitled toantedate such a disclosure by virtue of prior invention.

BRIEF DESCRIPTION OF THE DRAWINGS

This invention, as defined in the claims, can be better understood withreference to the following drawings. The drawings are not necessarily toscale, emphasis instead being placed upon clearly illustratingprinciples of the present invention.

FIG. 1: Clip-on model for saposin C induced fusion: Liposome-boundsaposin Cs clip one to another through hydrophobic interaction, andinduce liposome fusion.

FIG. 2: Saposin C and liposome vesicle association: A conformationalalteration of the saposin-fold found in lipid-bound saposin C. MembraneTopological interaction of saposin C indicated that amphipathic helicesat amino- and carboxyl-termini were embedded into the lipid bilayer andthe middle region of saposin C is exposed to aqueous phase. The middleregion of saposin C is exposed to the aqueous phase.

FIG. 3: A schematic of the functional organization of the neuritogenic,acid β-glucosidase activation and lipid-binding properties of saposin C.Except for the box indicating the predicated turn and the disulfidebonds, the figure is not meant to represent known physical structure.The residues from 22-32 are of major significance to the neurotrophiceffect. The region spanning residues 42-61 is critical to the acidβ-glucosidase activation effects of saposin C, and the presence of allthree disulfide bonds is also important for this function. In addition,higher order structure is required to have full activities of saposin C.Lipid/lipid membrane interaction regions are located at both NH₂- andCOOH-terminal regions.

FIG. 4: Size changes of BPS (brain phosphatidylserine) liposomes inducedby Ca²⁺ (a) or Saposin C (b) at pH 4.7 or 7.4. Fair autocorrelationfunction, dust=0.0%, base line error<1%, room temperature.

FIG. 5. Transport of NBD-DOPS and saposin C into cerebellum of mousebrains NBD-DOPS-saposin C proteoliposomes (A,C,D) and PBS (B,E,F) wereadministered through tail veins of FBV/N adult mice. Frozen cerebellumsections were prepared at 48 hours after injection. NBD greenfluorescence for detecting DOPS in (A) and (B) was visualized using amicroscope (Zeiss Axioskop, 100×). NBD green fluorescence (C and E) andanti-His antibody (a rhodamine-conjugated secondary antibody, redfluorescence) for detecting saposin C (D and F) in Purkinje cells wereimaged under a confocal microscope (LSM510, Zeiss). Bar: 20 μm (C-F).Terms: p=Purkinje cells; g=granular cells.

FIG. 6. Fluorescence Spectra for PTIR-271 (20 uM)/Saposin C-DOPSproteoliposomes in PBS (FIG. 7A), and PTIR-316 (20 μM)/SapC-DOPSproteoliposomes in PBS (FIG. 7B).

FIG. 7. Uptake of Saposin-C-DOPS containing PTIR-271 and PTIR-316 intohuman neuroblastoma cells (CHLA-20). FIGS. 8A and 8B show PTIR-271uptake as delivered by SapC-DOPS liposomes. FIGS. 8C and 8D showPTIR-316 uptake in as delivered by SapC-DOPS liposomes. Controlliposomes (treated with SapC-DOPS liposomes without PTIR-271 orPTIR-316) are shown in FIGS. 8E and 8F. Red images represent uptake ofdyes. Visualized using Zeiss Axiovert-ApoTome Microscope (63× and 40×oil lens): λ_(EX)/λ_(EM); Beam splitter: 660; B/W phase contrast forcell morphology. Axiovision software used for imaging.

FIG. 8. Delivery of GFP22 siRNA into EGFP 4T1 cells using Sap-C-DOPSliposomes. GFP22 siRNA is a 22 nucleotide double-stranded RNA thatspecifically inhibits green fluorescent protein gene expression. (NJCaplen et al., PNAS, 2001, 98:9742-9747). Incubation time was 72 hours.20×, 800 mSec exposure; Photoshop: input levels 27, 1.19, 164; outputlevel 255. Size 3×2.29 inches. FIGS. 9A and 9C represent GFP22 siRNAcontaining liposomes; FIGS. 9B and 9D represent negative controls inwhich a non-silencing siRNA (consisting of a 22 nucleotidedouble-stranded RNA fragment) that does not affect GFP expression wasused. All RNA was purchased from QIAGEN.

FIG. 9. Micrographs of the delivery of GFP 22 siRNA into neuroblastoma(CHLA-20) cancer cells showing (a) Rhodamine-GFP22 siRNA; (b)Phase-Contrast; and (c) merged.

In the following description of the illustrated embodiments, referencesare made to the accompanying drawings, which form a part hereof, and inwhich is shown by way of illustration various embodiments in which theinvention may be practiced. It is to be understood that otherembodiments may be utilized, and structural and functional changes maybe made without departing from the scope of the present invention.

DETAILED DESCRIPTION OF THE INVENTION

Before the present compositions and methods are described, it is to beunderstood that this invention is not limited to the specificmethodology, devices, and formulations as such may, of course, vary. Itis also to be understood that the terminology used herein is for thepurpose of describing particular embodiments only, and is not intendedto limit the scope of the present invention which will be limited onlyby the appended claims.

It must be noted that as used herein and in the appended claims, thesingular forms “a”, “and”, and “the” include plural referents unless thecontext clearly dictates otherwise. Unless defined otherwise, alltechnical and scientific terms used herein have the same meaning ascommonly understood to one of ordinary skill in the art to which thisinvention belongs. Although any methods, devices and materials similaror equivalent to those described herein can be used in the practice ortesting of the invention, the preferred methods, devices and materialsare now described.

Definitions

The terms “administered” and “administration” refer generally to theadministration to a patient of a biocompatible material, including, forexample, lipid and/or vesicle compositions and flush agents.Accordingly, “administered” and “administration” refer, for example, tothe injection into a blood vessel of lipid and/or vesicle compositionsand/or flush agents. The terms “administered” and “administration” canalso refer to the delivery of lipid and/or vesicle compositions and/orflush agents to a region of interest.

The terms “amino acid” or “amino acid sequence,” as used herein, referto an oligopeptide, peptide, polypeptide, or protein sequence, or afragment of any of these, and to naturally occurring or syntheticmolecules. Where “amino acid sequence” is recited herein to refer to anamino acid sequence of a naturally occurring protein molecule, “aminoacid sequence” and like terms are not meant to limit the amino acidsequence to the complete native amino acid sequence associated with therecited protein molecule.

The term “amphipathic lipid” means a molecule that has a hydrophilic“head” group and hydrophobic “tail” group and has membrane-formingcapability.

As used herein, the terms “anionic phospholipid membrane” and “anionicliposome” refer to a phospholipid membrane or liposome that containslipid components and has an overall negative charge at physiological pH.

“Anionic phospholipids” means phospholipids having negative charge,including phosphate, sulphate and glycerol-based lipids.

“Bioactive agent” refers to a substance which may be used in connectionwith an application that is therapeutic or diagnostic in nature, such asin methods for diagnosing the presence or absence of a disease in apatient and/or in methods for the treatment of disease in a patient. Asused herein, “bioactive agent” refers also to substances which arecapable of exerting a biological effect in vitro and/or in vivo. Thebioactive agents may be neutral or positively or negatively charged.Examples of suitable bioactive agents include diagnostic agents,pharmaceuticals, drugs, synthetic organic molecules, proteins, peptides,vitamins, steroids and genetic material, including nucleosides,nucleotides and polynucleotides.

The term “contained (with)in” refers to a pharmaceutical agent beingenveloped within a phospholipid membrane, such that the pharmaceuticalagent is protected from the outside environment. This term may be usedinterchangeably with “encapsulated.”

A “deletion,” as the term is used herein, refers to a change in theamino acid or nucleotide sequence that results in the absence of one ormore amino acid residues or nucleotides.

The term “derivative,” as used herein, refers to the chemicalmodification of a polypeptide sequence, or a polynucleotide sequence.Chemical modifications of a polynucleotide sequence can include, forexample, replacement of hydrogen by an alkyl, acyl, or amino group. Aderivative polynucleotide encodes a polypeptide which retains at leastone biological function of the natural molecule. A derivativepolypeptide is one modified, for instance by glycosylation, or any otherprocess which retains at least one biological function of thepolypeptide from which it was derived.

The term “fusogenic protein or polypeptide” as used herein refers to aprotein or peptide that when added to two separate bilayer membranes canbring about their fusion into a single membrane. The fusogenic proteinforces the cell or model membranes into close contact and causes them tofuse.

The words “insertion” or “addition,” as used herein, refer to changes inan amino acid or nucleotide sequence resulting in the addition of one ormore amino acid residues or nucleotides, respectively, to the sequencefound in the naturally occurring molecule.

The terms “lipid” and “phospholipid” are used interchangeably and torefer to structures containing lipids, phospholipids, or derivativesthereof comprising a variety of different structural arrangements whichlipids are known to adopt in aqueous suspension. These structuresinclude, but are not limited to, lipid bilayer vesicles, micelles,liposomes, emulsions, vesicles, lipid ribbons or sheets. In thepreferred embodiment, the lipid is an anionic liposome. The lipids maybe used alone or in any combination which one skilled in the art wouldappreciate to provide the characteristics desired for a particularapplication. In addition, the technical aspects of lipid constructs andliposome formation are well known in the art and any of the methodscommonly practiced in the field may be used for the present invention.

“Lipid composition” refers to a composition which comprises a lipidcompound, typically in an aqueous medium. Exemplary lipid compositionsinclude suspensions, emulsions and vesicle compositions. “Lipidformulation” refers to a lipid composition which also comprises abioactive agent.

“Liposome” refers to a generally spherical cluster or aggregate ofamphipathic compounds, including lipid compounds, typically in the formof one or more concentric layers, for example, bilayers. They may alsobe referred to herein as lipid vesicles.

The term “long-chain lipid” refers to lipids having a carbon chainlength of about 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or 24. In oneembodiment, the chain length is selected from a chain length of 18, 19,or 20. Examples of lipids that may be used with the present inventionare available on the website www.avantilipids.com. Representativeexamples of long chain lipids that may be used with the presentinvention include, but are not limited to the following lipids:

-   14:0 PS 1,2-Dimyristoyl-sn-Glycero-3-[Phospho-L-Serine] (Sodium    Salt) (DMPS); 16:0 PS    1,2-Dipalmitoyl-sn-Glycero-3-[Phospho-L-Serine] (Sodium Salt)    (DPPS); 17:0 PS 1,2-Diheptadecanoyl-sn-Glycero-3-[Phospho-L-Serine]    (Sodium Salt); 18:0 PS    1,2-Distearoyl-sn-Glycero-3-[Phospho-L-Serine] (Sodium Salt) (DSPS);    18:1 PS 1,2-Dioleoyl-sn-Glycero-3-[Phospho-L-Serine] (Sodium Salt)    (DOPS); 18:2 PS 1,2-Dilinoleoyl-sn-Glycero-3-[Phospho-L-Serine]    (Sodium Salt); 20:4 PS    1,2-Diarachidonoyl-sn-Glycero-3-[Phospho-L-Serine] (Sodium Salt);    22:6 PS 1,2-Didocosahexaenoyl-sn-Glycero-3-[Phospho-L-Serine]    (Sodium Salt); 16:0-18:1 PS    1-Palmitoyl-2-Oleoyl-sn-Glycero-3-[Phospho-L-Serine] (Sodium Salt)    (POPS); 16:0-18:2 PS    1-Palmitoyl-2-Linoleoyl-sn-Glycero-3-[Phospho-L-Serine] (Sodium    Salt); 16:0-22:6 PS    1-Palmitoyl-2-Docosahexaenoyl-sn-Glycero-3-[Phospho-L-Serine]    (Sodium Salt); 18:0-18:1 PS    1-Stearoyl-2-Oleoyl-sn-Glycero-3-[Phospho-L-Serine] (Sodium Salt);    18:0-18:2 PS 1-Stearoyl-2-Linoleoyl-sn-Glycero-3-[Phospho-L-Serine]    (Sodium Salt); 18:0-20:4 PS    1-Stearoyl-2-Arachidonoyl-sn-Glycero-3-[Phospho-L-Serine] (Sodium    Salt); 18:0-22:6 PS    1-Stearoyl-2-Docosahexaenoyl-sn-Glycero-3-[Phospho-L-Serine] (Sodium    Salt); 16:0 PC 1,2-Dipalmitoyl-sn-Glycero-3-Phosphocholine (DPPC);    17:0 PC 1,2-Diheptadecanoyl-sn-Glycero-3-Phosphocholine; 18:0 PC    1,2-Distearoyl-sn-Glycero-3-Phosphocholine (DSPC); 16:1 PC (Cis)    1,2-Dipalmitoleoyl-sn-Glycero-3-Phosphocholine; 16:1 Trans PC    1,2-Dipalmitelaidoyl-sn-Glycero-3-Phosphocholine; 18:1 PC Delta6    (cis) 1,2-Dipetroselinoyl-sn-Glycero-3-Phosphocholine; 18:2 PC (cis)    1,2-Dilinoleoyl-sn-Glycero-3-Phosphocholine; 18:3 PC (cis)    1,2-Dilinolenoyl-sn-Glycero-3-Phosphocholine; 20:1 PC (cis)    1,2-Dieicosenoyl-sn-Glycero-3-Phosphocholine; 22:1 PC (cis)    1,2-Dierucoyl-sn-Glycero-3-Phosphocholine; 22:0 PC    1,2-Dibehenoyl-sn-Glycero-3-Phosphocholine; 24:1 PC (cis)    1,2-Dinervonoyl-sn-Glycero-3-Phosphocholine; 16:0-18:0 PC    1-Palmitoyl-2-Stearoyl-sn-Glycero-3-Phosphocholine; 16:0-18:1 PC    1-Palmitoyl-2-Oleoyl-sn-Glycero-3-Phosphocholine; 16:0-18:2 PC    1-Palmitoyl-2-Linoleoyl-sn-Glycero-3-Phosphocholine; 18:0-18:1 PC    1-Stearoyl-2-Oleoyl-sn-Glycero-3-Phosphocholine; 18:0-18:2 PC    1-Stearoyl-2-Linoleoyl-sn-Glycero-3-Phosphocholine; 18:1-18:0 PC    1-Oleoyl-2-Stearoyl-sn-Glycero-3-Phosphocholine; 18:1-16:0 PC    1-Oleoyl-2-Palmitoyl-sn-Glycero-3-Phosphocholine; 18:0-20:4 PC    1-Stearoyl-2-Arachidonyl-sn-Glycero-3-Phosphocholine; 16:0-18:1 PG    1-Palmitoyl-2-Oleoyl-sn-Glycero-3-[Phospho-rac-(1-glycerol)] (Sodium    Salt) (POPG); 18:1 PG    1,2-Dioleoyl-sn-Glycero-3-[Phospho-rac-(1-glycerol)] (Sodium Salt)    (DOPG); 18:1 PA 1,2-Dioleoyl-sn-Glycero-3-Phosphate (Monosodium    Salt) (DOPA); 18:1 PI 1,2-Dioleoyl-sn-Glycero-3-Phosphoinositol    (Ammonium Salt); 16:0(D31)-18:1 PI    1-Palmitoyl(D31)-2-Oleoyl-sn-Glycero-3-Phosphoinositol (Ammonium    Salt); 18:1 PE 1,2-Dioleoyl-sn-Glycero-3-Phosphoethanolamine (DOPE);    18:2 PE 1,2-Dilinoleoyl-sn-Glycero-3-Phosphoethanolamine.

The phrases “nucleic acid” or “nucleic acid sequence,” as used herein,refer to a nucleotide, oligonucleotide, polynucleotide, or any fragmentthereof. A “nucleic acid” refers to a string of at least twobase-sugar-phosphate combinations. (A polynucleotide is distinguishedfrom an oligonucleotide by containing more than 120 monomeric units.)Nucleotides are the monomeric units of nucleic acid polymers. The termincludes deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) in theform of an oligonucleotide messenger RNA, anti-sense, plasmid DNA, partsof a plasmid DNA or genetic material derived from a virus. Anti-sense isa polynucleotide that interferes with the function of DNA and/or RNA.The term nucleic acid refers to a string of at least twobase-sugar-phosphate combinations. Natural nucleic acids have aphosphate backbone, artificial nucleic acids may contain other types ofbackbones, but contain the same bases. Nucleotides are the monomericunits of nucleic acid polymers. The term includes deoxyribonucleic acid(DNA) and ribonucleic acid (RNA). RNA may be in the form of a tRNA(transfer RNA), siRNA (short interfering ribonucleic acid), snRNA (smallnuclear RNA), rRNA (ribosomal RNA), mRNA (messenger RNA), anti-senseRNA, and ribozymes. DNA may be in form plasmid DNA, viral DNA, linearDNA, or chromosomal DNA or derivatives of these groups. In additionthese forms of DNA and RNA may be single, double, triple, or quadruplestranded. The term also includes PNAs (peptide nucleic acids), siNA(short interfering nucleic acid), phosphorothioates, and other variantsof the phosphate backbone of native nucleic acids.

As used herein, the term “nucleotide-based pharmaceutical agent” or“nucleotide-based drug” refer to a pharmaceutical agent or drugcomprising a nucleotide, an oligonucleotide or a nucleic acid. \

“Patient” or “subject” refers to animals, including mammals, preferablyhumans.

As used herein, “pharmaceutical agent or drug” refers to any chemical orbiological material, compound, or composition capable of inducing adesired therapeutic effect when properly administered to a patient. Somedrugs are sold in an inactive form that is converted in vivo into ametabolite with pharmaceutical activity. For purposes of the presentinvention, the terms “pharmaceutical agent” and “drug” encompass boththe inactive drug and the active metabolite.

The phrase “pharmaceutically or therapeutically effective dose oramount” refers to a dosage level sufficient to induce a desiredbiological result. That result may be the delivery of a pharmaceuticalagent, alleviation of the signs, symptoms or causes of a disease or anyother desired alteration of a biological system and the precise amountof the active depends on the physical condition of the patient,progression of the illness being treated etc.

As used herein, the term “saposin” refers to the family ofprosaposin-derived proteins and polypeptides, including but not limitedto naturally occurring saposins A, B, C and D as well as syntheticsaposin-derived proteins and peptides and peptide analogs showingfusogenic activity. The saposin C and polypeptides derived therefrom maybe used in one embodiment of the invention.

The term “short chain lipid” refers to lipids having a carbon chainlength of 4, 5, 6, 7, 8, 9, 10, 11 or 12. In one embodiment, the carbonchain length is 6, 7, 8 9 or 10. In one embodiment, the carbon chainlength is 6, 7 or 8. Examples of negative short chain lipids areavailable at the website www.avantilipids.com. Examples of short chainlipids that may be used with the present invention include, but are notlimited to, the following: 06:0 PS (DHPS)1,2-Dihexanoyl-sn-Glycero-3-[Phospho-L-Serine] (Sodium Salt); 08:0 PS1,2-Dioctanoyl-sn-Glycero-3-[Phospho-L-Serine] (Sodium Salt); 03:0 PC1,2-Dipropionoyl-sn-Glycero-3-Phosphocholine; 04:0 PC1,2-Dibutyroyl-sn-Glycero-3-Phosphocholine; 05:0 PC1,2-Divaleroyl-sn-Glycero-3-Phosphocholine; 06:0 PC (DHPC)1,2-Dihexanoyl-sn-Glycero-3-Phosphocholine; 07:0 PC1,2-Diheptanoyl-sn-Glycero-3-Phosphocholine; 08:0 PC1,2-Dioctanoyl-sn-Glycero-3-Phosphocholine; 09:0 PC1,2-Dinonanoyl-sn-Glycero-3-Phosphocholine; 06:0 PG1,2-Dihexanoyl-sn-Glycero-3-[Phospho-rac-(1-glycerol)] (Sodium Salt);08:0 PG 1,2-Dioctanoyl-sn-Glycero-3-[Phospho-rac-(1-glycerol)] (SodiumSalt); 06:0 PA 1,2-Dihexanoyl-sn-Glycero-3-Phosphate (Monosodium Salt);08:0 PA 1,2-Dioctanoyl-sn-Glycero-3-Phosphate (Monosodium Salt); 06:0 PE1,2-Dihexanoyl-sn-Glycero-3-Phosphoethanolamine; 08:0 PE1,2-Dioctanoyl-sn-Glycero-3-Phosphoethanolamine.

As used herein, the term “short interfering nucleic acid”, “siNA”,“short interfering RNA”, “siRNA”, “short interfering nucleic acidmolecule”, “short interfering oligonucleotide molecule”, or“chemically-modified short interfering nucleic acid molecule”, refers toany nucleic acid molecule capable of inhibiting or down regulating geneexpression or viral replication, for example by mediating RNAinterference “RNAi” or gene silencing in a sequence-specific manner.Within exemplary embodiments, the siNA is a double-strandedpolynucleotide molecule comprising self-complementary sense andantisense regions, wherein the antisense region comprises a nucleotidesequence that is complementary to a nucleotide sequence in a targetnucleic acid molecule for down regulating expression, or a portionthereof, and the sense region comprises a nucleotide sequencecorresponding to (i.e., which is substantially identical in sequence to)the target nucleic acid sequence or portion thereof. “siNA” means asmall interfering nucleic acid, for example a siRNA, that is ashort-length double-stranded nucleic acid (or optionally a longerprecursor thereof), and which is not unacceptably toxic in target cells.The length of useful siNAs within the invention will in certainembodiments be optimized at a length of approximately 21 to 23 bp long.However, there is no particular limitation in the length of usefulsiNAs, including siRNAs. For example, siNAs can initially be presentedto cells in a precursor form that is substantially different than afinal or processed form of the siNA that will exist and exert genesilencing activity upon delivery, or after delivery, to the target cell.Precursor forms of siNAs may, for example, include precursor sequenceelements that are processed, degraded, altered, or cleaved at orfollowing the time of delivery to yield a siNA that is active within thecell to mediate gene silencing. Thus, in certain embodiments, usefulsiNAs within the invention will have a precursor length, for example, ofapproximately 100-200 base pairs, 50-100 base pairs, or less than about50 base pairs, which will yield an active, processed siNA within thetarget cell. In other embodiments, a useful siNA or siNA precursor willbe approximately 10 to 49 bp, 15 to 35 bp, or about 21 to 30 bp inlength.

“Vesicle” refers to a spherical entity which is generally characterizedby the presence of one or more walls or membranes which form one or moreinternal voids. Vesicles may be formulated, for example, from lipids,including the various lipids described herein, proteinaceous materials,polymeric materials, including natural, synthetic and semi-syntheticpolymers, or surfactants. Preferred vesicles are those which comprisewalls or membranes formulated from lipids. In these preferred vesicles,the lipids may be in the form of a monolayer or bilayer, and the mono-or bilayer lipids may be used to form one or more mono- or bilayers. Inthe case of more than one mono- or bilayer, the mono- or bilayers may beconcentric. Lipids may be used to form a unilamellar vesicle (comprisedof one monolayer or bilayer), an oligolamellar vesicle (comprised ofabout two or about three monolayers or bilayers) or a multilamellarvesicle (comprised of more than about three monolayers or bilayers).Similarly, the vesicles prepared from proteins or polymers may compriseone or more concentric walls or membranes. The walls or membranes ofvesicles prepared from proteins or polymers may be substantially solid(uniform), or they may be porous or semi-porous. The vesicles describedherein include such entities commonly referred to as, for example,liposomes, micelles, bubbles, microbubbles, microspheres, lipid-,polymer-protein- and/or surfactant-coated bubbles, microbubbles and/ormicrospheres, microballoons, aerogels, clathrate bound vesicles, and thelike. The internal void of the vesicles may be filled with a liquid(including, for example, an aqueous liquid), a gas, a gaseous precursor,and/or a solid or solute material, including, for example, a targetingligand and/or a bioactive agent, as desired.

Fusogenic Proteins or Polypeptides

In one embodiment, the present invention provides for phopholipidmembranes comprising one or more lysosomal fusogenic protein orpolypeptide. In another embodiment, the one or more lysosomal fusogenicprotein or polypeptide is contained within anionic liposomes. In anotherembodiment, the anionic liposomes further comprise a pharmaceuticalagent.

Suitable lysosomal fusogenic proteins and polypeptides for use in thisinvention include, but are not limited to, proteins of the saposinfamily, for example, saposin C. Also included are homologues of saposinC, wherein the homologue possesses at least 80% sequence homology, dueto degeneracy of the genetic code which encodes for saposin C, andpolypeptides and peptide analogues possessing similar biologicalactivity as saposin C.

Examples of peptides or peptide analogues include:

(SEQ. ID. No. 1) Ser-Asp-Val-Tyr-Cys-Glu-Val-Cys-Glu-Phe-Leu-Val-Lys-Glu-Val-Thr-Lys-Leu-Ile-Asp-Asn-Asn-Lys-Thr-Glu-Lys-Glu-Ile-Leu-Asp-Ala-Phe-Asp-Lys-Met-Cys- Ser-Lys-Leu-Pro;(SEQ. ID. No. 2) Val-Tyr-Cys-Glu-Val-Cys-Glu-Phe-Leu-Val-Lys-Glu-Val-Thr-Lys-Leu-Ile-Asp-Asn-Asn-Lys-Thr-Glu-Lys-Glu-Ile-Leu-Asp-Ala-Phe-Asp-Lys-Met-Cys-Ser-Lys- Leu-Pro,

and derivatives, analogues, homologues, fragments and mixtures thereof.

Also included are polypeptides of the formula:

h-u-Cys-Glu-h-Cys-Glu-h-h-h-Lys-Glu-h-u-Lys-h-h-Asp-Asn-Asn-Lys-u-Glu-Lys-Glu-h-h-Asp-h-h-Asp- Lys-h-Cys-u-Lys-h-h,

-   -   where h=hydrophobic amino acids, including, Val, Leu, Ile, Met,        Pro, Phe, and Ala; and u=uncharged polar amino acids, including,        Thr, Ser, Tyr, Gly, Gln, and Asn.

Suitable lysosomal fusogenic proteins and polypeptides for use in thisinvention include, but are not limited to, proteins of the saposinfamily, preferably saposin C. Also included are homologues of saposin C,wherein the homologue possesses at least 80% sequence homology, due todegeneracy of the genetic code which encodes for saposin C, andpolypeptides and peptide analogues possessing similar biologicalactivity as saposin C.

As used herein, term “peptide analog” refers to a peptide which differsin amino acid sequence from the native peptide only by conservativeamino acid substitutions, for example, substitution of Leu for Val, orArg for Lys, etc., or by one or more non-conservative amino acidsubstitutions, deletions, or insertions located at positions which donot destroy the biological activity of the peptide (in this case, thefusogenic property of the peptide). A peptide analog, as used herein,may also include, as part or all of its sequence, one or more amino acidanalogues, molecules which mimic the structure of amino acids, and/ornatural amino acids found in molecules other than peptide or peptideanalogues.

By “analogs” is meant substitutions or alterations in the amino acidsequences of the peptides of the invention, which substitutions oralterations do not adversely affect the fusogenic properties of thepeptides. Thus, an analog might comprise a peptide having asubstantially identical amino acid sequence to a peptide provided hereinas SEQ ID NO:1 and 2 and in which one or more amino acid residues havebeen conservatively substituted with chemically similar amino acids.Examples of conservative substitutions include the substitution of anon-polar (hydrophobic) residue such as isoleucine, valine, leucine ormethionine for another. Likewise, the present invention contemplates thesubstitution of one polar (hydrophilic) residue such as between arginineand lysine, between glutamine and asparagine, and between glycine andserine. Additionally, the substitution of a basic residue such aslysine, arginine or histidine for another or the substitution of oneacidic residue such as aspartic acid or glutamic acid for another isalso contemplated.

Phospholipid Membrane and Formation of Liposomes

This invention utilizes an anionic phospholipid membrane to effect thesaposin-mediated membrane fusion for delivery of a particularpharmaceutical or imaging agent across either a dermal or mucosalmembrane or across the blood-brain barrier or other cellular membranes.These anionic phospholipid membranes are generally used for preparingliposomes. Liposomes are microscopic vesicles consisting of concentriclipid bilayers and, as used herein, refer to small vesicles composed ofamphipathic lipids arranged in spherical bilayers. Structurally,liposomes range in size and shape from long tubes to spheres, withdimensions from a few hundred angstroms to fractions of a millimeter.Regardless of the overall shape, the bilayers are generally organized asclosed concentric lamellae, with an aqueous layer separating eachlamella from its neighbor. Vesicle size normally falls in a range ofbetween about 20 and about 30,000 nm in diameter.

The liquid film between lamellae is usually between about 3 and 10 nm. Avariety of methods for preparing various liposome forms have beendescribed in the periodical and patent literature. For specific reviewsand information on liposome formulations, reference is made to reviewsby Pagano and Weinstein (see Ann. Rev. Biophysic. Bioeng., 7, 435-68(1978) and Ann. Rev. Biophysic. Bioeng., 9, 467-508 (1980)).

In one embodiment, the anionic phospholipid membrane is a vesicle. Inanother embodiment, the vesicle is a liposome. The liposomes are a formof nanocontainer and nanocontainers, such as nanoparticles or liposomes,are commonly used for encapsulation of drugs. The liposomes preferablyhave mean diameters of about 200 nanometers. In another embodiment, theliposomes have a mean diameter of between 50 and 350 nanometers. Inanother embodiment, the liposomes have a mean diameter of between 150and 250 nanometers.

Specific delivery of liposomes to a target tissue such as aproliferating cell mass, neoplastic tissue, inflammatory tissue,inflamed tissue, and infected tissue can be achieved by selecting aliposome size appropriate for delivering a therapeutic agent to saidtarget tissue. For example, liposomes with a mean diameter of 180 nm maynot accumulate in a solid tumor; liposomes with a mean diameter of 140nm accumulate in the periphery of the same solid tumor, and liposomeswith a mean diameter of 110 nm accumulate in the peripheral and centralportions of that solid tumor.

In connection with embodiments involving vesicle compositions, the sizeof the vesicles can be adjusted for the particular intended end useincluding, for example, diagnostic and/or therapeutic use. The size ofthe vesicles may preferably range from about 30 nanometers (nm) to about300 micrometers (μm) in diameter, and all combinations andsubcombinations of ranges therein. More preferably, the vesicles havediameters of from about 100 nm to about 10 μm, with mean diameters offrom about 200 nm to about 7 μm being even more preferred. In connectionwith particular uses, for example, intravascular use, including magneticresonance imaging of the vasculature, it may be preferred that thevesicles be no larger that about 30 μm in diameter, with smallervesicles being preferred, for example, vesicles of no larger than about12 μm in diameter. In certain preferred embodiments, the diameter of thevesicles may be about 7 μm or less, with vesicles having a mean diameterof about 5 μm or less being more preferred, and vesicles having a meandiameter of about 3 μm or less being even more preferred.

The size of the liposomes can be adjusted, if desired, by a variety ofprocedures including, for example, shaking, microemulsification,vortexing, extrusion, filtration, sonication, homogenization, repeatedfreezing and thawing cycles, extrusion under pressure through pores ofdefined size, and similar methods.

In addition to, or instead of, the lipid, proteinaceous and/or polymericcompounds discussed above, the compositions described herein maycomprise one or more stabilizing materials. Exemplary of suchstabilizing materials are, for example, biocompatible polymers. Thestabilizing materials may be employed to desirably assist in theformation of vesicles and/or to assure substantial encapsulation of thegases or gaseous precursors. Even for relatively insoluble,non-diffusible gases, such as perfluoropropane or sulfur hexafluoride,improved vesicle compositions may be obtained when one or morestabilizing materials are utilized in the formation of the gas andgaseous precursor filled vesicles. These compounds may help improve thestability and the integrity of the vesicles with regard to their size,shape and/or other attributes.

The terms “stable” or “stabilized”, as used herein, means that thevesicles may be substantially resistant to degradation, including, forexample, loss of vesicle structure or encapsulated gas or gaseousprecursor, for a useful period of time. Typically, the vesicles employedin the present invention have a desirable shelf life, often retaining atleast about 90% by volume of its original structure for a period of atleast about two to three weeks under normal ambient conditions. Inpreferred form, the vesicles are desirably stable for a period of timeof at least about 1 month, more preferably at least about 2 months, evenmore preferably at least about 6 months, still more preferably abouteighteen months, and yet more preferably up to about 3 years. Thevesicles described herein, including gas and gaseous precursor filledvesicles, may also be stable even under adverse conditions, such astemperatures and pressures which are above or below those experiencedunder normal ambient conditions.

The stability of the vesicles described herein may be attributable, atleast in part, to the materials from which the vesicles are made,including, for example, the lipids, polymers and/or proteins describedabove, and it is often not necessary to employ additional stabilizingmaterials, although it is optional and may be preferred to do so. Suchadditional stabilizing materials and their characteristics are describedmore fully hereinafter.

The materials from which the vesicles are constructed are preferablybiocompatible lipid, protein or polymer materials, and of these, thebiocompatible lipids are preferred. In addition, because of the ease offormulation, including the capability of preparing vesicles immediatelyprior to administration, these vesicles may be conveniently made onsite.

The biocompatible polymers useful as stabilizing materials for preparingthe gas and gaseous precursor filled vesicles may be of natural,semi-synthetic (modified natural) or synthetic origin. As used herein,the term polymer denotes a compound comprised of two or more repeatingmonomeric units, and preferably 10 or more repeating monomeric units.The phrase semi-synthetic polymer (or modified natural polymer), asemployed herein, denotes a natural polymer that has been chemicallymodified in some fashion. Exemplary natural polymers suitable for use inthe present invention include naturally occurring polysaccharides. Suchpolysaccharides include, for example, arabinans, fructans, fucans,galactans, galacturonans, glucans, mannans, xylans (such as, forexample, inulin), levan, fucoidan, carrageenan, galatocarolose, pecticacid, pectins, including amylose, pullulan, glycogen, amylopectin,cellulose, dextran, dextrin, dextrose, polydextrose, pustulan, chitin,agarose, keratan, chondroitan, dermatan, hyaluronic acid, alginic acid,xanthan gum, starch and various other natural homopolymer orheteropolymers, such as those containing one or more of the followingaldoses, ketoses, acids or amines: erythrose, threose, ribose,arabinose, xylose, lyxose, allose, altrose, lucose, mannose, gulose,idose, galactose, talose, erytirulose, ribulose, xylulose, psicose,fructose, sorbose, tagatose, mannitol, sorbitol, lactose, sucrose,trehalose, maltose, cellobiose, glycine, serine, threonine, cysteine,tyrosine, asparagine, glutamine, aspartic acid, glutamic acid, lysine,arginine, histidine, glucuronic acid, gluconic acid, glucaric acid,galacturonic acid, mannuronic acid, glucosamine, galactosamine, andneuraminic acid, and naturally occurring derivatives thereofAccordingly, suitable polymers include, for example, proteins, such asalbumin. Exemplary semi-synthetic polymers includecarboxymethylcellulose, hydroxymethylcellulose,hydroxypropylmethylcellulose, methylcellulose, and methoxycellulose.Exemplary synthetic polymers suitable for use in the present inventioninclude polyethylenes (such as, for example, polyethylene glycol,polyoxyethylene, and polyethylene terephthlate), polypropylenes (suchas, for example, polypropylene glycol), polyurethanes (such as, forexample, polyvinyl alcohol (PVA), polyvinyl chloride andpolyvinylpyrrolidone), polyamides including nylon, polystyrene,polylactic acids, fluorinated hydrocarbons, fluorinated carbons (suchas, for example, polytetrafluoroethylene), and polymethylmethacrylate,and derivatives thereof. Methods for the preparation of vesicles whichemploy polymers as stabilizing compounds will be readily apparent tothose skilled in the art, once armed with the present disclosure, whenthe present disclosure is coupled with information known in the art,such as that described and referred to in U.S. Pat. No. 5,205,290, thedisclosures of which are hereby incorporated herein by reference, intheir entirety.

In general, the liposomes utilized in the present invention can bedivided into three categories based on their overall size and the natureof the lamellar structure (see New York Academy Sciences Meeting,“Liposomes and Their Use in Biology and Medicine,” of December 1977).The four classifications include multi-lamellar vesicles (MLV's), smalluni-lamellar vesicles (SUV's), large uni-lamellar vesicles (LUV's) andgiant unilamellar vesicles (GUV's). SUVs and LUVs, by definition, haveonly one bilayer, whereas MLVs contain many concentric bilayers.Spherical unilamellar vesicles (ULV) with a low polydispersity canspontaneously form in charged phospholipid mixtures. The formation ofsuch low-polydispersity ULV usually requires a process of abruptincrease in temperature or sudden dilution. In some cases, thespontaneous low-polydispersity ULVs have been examined to be highlystable over time and upon dilution, which illustrates a great potentialto be encapsulating carriers for drug delivery or gene therapy. SeeNieh, et al. Low-Polydispersity Phospholipid Unilamellar EllipsoidalVesicles and Their Interaction with Helical Domains of Saposin C.,Manuscript.

Liposomes exhibit a wide variety of characteristics, depending upontheir size, composition, and charge. For example, liposomes having asmall percentage of unsaturated lipids tend to be slightly morepermeable, while liposomes incorporating cholesterol or other sterolstend to be more rigid and less permeable. Liposomes may be positive,negative, or neutral in charge, depending on the hydrophilic group. Forexample, choline-based lipids impart an overall neutral charge,phosphate and sulfate based lipids contribute a negative charge,glycerol-based lipids are generally negatively-charged, and sterols aregenerally neutral in solution but have charged groups. The lipids usedin the present invention are both anionic and neutral lipids.

A wide variety of methods are available in connection with thepreparation of liposome compositions. Accordingly, the liposomes may beprepared using any one of a variety of conventional liposomalpreparatory techniques which will be apparent to those skilled in theart. These techniques include, for example, solvent dialysis, Frenchpress, extrusion (with or without freeze-thaw), reverse phaseevaporation, simple freeze-thaw, sonication, chelate dialysis,homogenization, solvent infusion, microemulsification, spontaneousformation, solvent vaporization, solvent dialysis, French pressure celltechnique, controlled detergent dialysis, and others, each involving thepreparation of the vesicles in various fashions. See, e.g., Madden etal., Chemistry and Physics of Lipids, 1990 53, 37-46, the disclosures ofwhich are hereby incorporated herein by reference in their entirety.Suitable freeze-thaw techniques are described, for example, inInternational Application Ser. No. PCT/US89/05040, filed Nov. 8, 1989,the disclosures of which are incorporated herein by reference in theirentirety. Methods which involve freeze-thaw techniques are preferred inconnection with the preparation of liposomes. Preparation of theliposomes may be carried out in a solution, such as an aqueous salinesolution, aqueous phosphate buffer solution, or sterile water. Theliposomes may also be prepared by various processes which involveshaking or vortexing. This may be achieved, for example, by the use of amechanical shaking device, such as a Wig-L-Bug (Crescent Dental, Lyons,Ill.), a Mixomat, sold by Degussa AG, Frankfurt, Germany, a Capmix, soldby Espe Fabrik Pharmazeutischer Praeparate GMBH & Co., Seefeld, OberayGermany, a Silamat Plus, sold by Vivadent, Lechtenstein, or a Vibros,sold by Quayle Dental, Sussex, England. Conventional microemulsificationequipment, such as a Microfluidizer(Microfluidics, Woburn, Mass.) mayalso be used.

Spray drying may be also employed to prepare the vesicles. Utilizingthis procedure, the lipids may be pre-mixed in an aqueous environmentand then spray dried to produce gas-filled vesicles. The vesicles may bestored under a headspace of a desired gas.

Many liposomal preparatory techniques which may be adapted for use inthe preparation of vesicle compositions are discussed, for example, inU.S. Pat. No. 4,728,578; U.K. Patent Application GB 2193095 A; U.S. Pat.No. 4,728,575; U.S. Pat. No. 4,737,323; International Application Ser.No. PCT/US85/01161; Mayer et al., Biochimica et Biophysica Acta, Vol.858, pp. 161-168 (1986); Hope et al., Biochimica et Biophysica Acta,Vol. 812, pp. 55-65 (1985); U.S. Pat. No. 4,533,254; Mayhew et al.,Methods in Enzymology, Vol. 149, pp. 64-77 (1987); Mayhew et al.,Biochimica et Biophysica Acta, Vol 755, pp. 169-74 (1984); Cheng et al,Investigative Radiology, Vol. 22, pp. 47-55 (1987); InternationalApplication Ser. No. PCT/US89/05040; U.S. Pat. No. 4,162,282; U.S. Pat.No. 4,310,505; U.S. Pat. No. 4,921,706; and Liposome Technology,Gregoriadis, G., ed., Vol. I, pp. 29-31, 51-67 and 79-108 (CRC PressInc., Boca Raton, Fla. 1984), the disclosures of each of which arehereby incorporated by reference herein, in their entirety.

Alternatively, one or more anti-bactericidal agents and/or preservativesmay be included in the formulation of the compositions, such as sodiumbenzoate, quaternary ammonium salts, sodium azide, methyl paraben,propyl paraben, sorbic acid, ascorbylpalmitate, butylatedhydroxyanisole, butylated hydroxytoluene, chlorobutanol, dehydroaceticacid, ethylenediamine, monothioglycerol, potassium benzoate, potassiummetabisulfite, potassium sorbate, sodium bisulfite, sulfur dioxide, andiorganic mercurial salts. Such sterilization, which may also be achievedby other conventional means, such as by irradiation, will be necessarywhere the stabilized vesicles are used for imaging under invasivecircumstances, e.g., intravascularly or intraperitonealy. Theappropriate means of sterilization will be apparent to the artisan basedon the present disclosure.

As with the preparation of lipid and/or vesicle compositions, a widevariety of techniques are available for the preparation of lipidformulations. For example, the lipid and/or vesicle formulations may beprepared from a mixture of lipid compounds, protein and bioactiveagents. In this case, lipid compositions are prepared as described abovein which the compositions also comprise bioactive agent. Thus, forexample, micelles can be prepared in the presence of a bioactive agent.

As those skilled in the art will recognize, any of the lipid and/orvesicle compositions and/or lipid and/or vesicle formulations may belyophilized for storage, and reconstituted, for example, with an aqueousmedium (such as sterile water, phosphate buffered solution, or aqueoussaline solution), with the aid of vigorous agitation. To preventagglutination or fusion of the lipids and/or vesicles as a result oflyophilization, it may be useful to include additives which prevent suchfusion or agglutination from occurring. Additives which may be usefulinclude sorbitol, mannitol, sodium chloride, glucose, trehalose,polyvinylpyrrolidone and poly(ethylene glycol) (PEG), for example, PEG400. These and other additives are described in the literature, such asin the U.S. Pharmacopeia, USP XXII, NF XVII, The United StatesPharmacopeia, The National Formulary, United States PharmacopeialConvention Inc., 12601 Twinbrook Parkway, Rockville, Md. 20852, thedisclosures of which are hereby incorporated herein by reference intheir entirety. Lyophilized preparations generally have the advantage ofgreater shelf life.

In general, the lipid mixtures of the present invention are comprised ofanionic long-chain lipids. In one embodiment, the lipid mixture used tosynthesize saposin C-containing liposomes is comprised of 1) anioniclong-chain lipids, 2) neutral long-chain lipids, and 3) short-chainlipids. The short chain lipids may be either neutral or anionic. Inanother embodiment, the lipid mixture is comprised only of anioniclong-chain lipids and neutral or anionic short-chain lipids. Thefollowing table illustrates examples of combinations of phospholipidsthat may be used to synthesize liposomes containing saposin C such thatthe objects of the present invention are achieved. Saposin C or apolypeptide of Saposin C may be added to the following combinations oflipids using the methods described herein. The following Table 1illustrates combinations of phospholipids that may be used to practicethe present invention. The examples are not exhaustive, but are intendedto illustrate possible embodiments of the present invention.

TABLE 1 Table 1. Combinations of Long Chain and Short ChainPhospholipids that may be used in combination with Saposin C orpolypeptides of Saposin C to form fusogenic-protein-containing Liposomesin Accordance with the Present Invention. Long-Chain PhospholipidShort-Chain Phospholipid 18:1 PS 18:0 PC 06:0 PC (DHPC) 18:1 PS 06:0 PC(DHPC) 18:1 PS 18:0 PC 06:0 PS (DHPS) 18:1 PS 06:0 PS (DHPS) 18:2 PS18:1 PG 06:0 PS (DHPS) 18:0-18:1 PS 18:1 PE 06:0 PC(DHPC) 16:0 PS 16:1PC 05:0 PC 20:4 PS 20:1 PC 07:0 PC

The presence of saposin C protein in the liposomal complex has beenobserved to destabilize and restructure the liposomal membrane,resulting in a limited shelf life for liposomal delivery systemsutilizing this protein. See Mu-Ping Nieh et al., Low-PolydispersityPhospholipid Unilamellar Ellipsoidal Vesicles and Their Interaction withHelical Domains of Saposin C; 2005. The present invention addresses thisproblem. One embodiment of the present invention includes the use of atleast one type of short-chain lipid. Addition of a short-chain lipidresults in stabilization of the membrane and an increase in liposomeshelf-life, increasing the utility and availability of liposomal-basedtherapeutics.

One example of a lipid mixture used to synthesize saposin-C liposomes isone that includes the negatively charged lipiddioleoylphosphatidylserine (DOPS) wherein a small amount of the neutrallong chain lipid dipalmitoyl phosphatidylcholine (DPPC) and the neutralshort-chain lipid dihexanoyl phosphatidycholine (DHPC) is added. See forexample, Nieh et al., Low-Polydispersity Phospholipid UnilamellarEllipsoidal Vesicles and Their Interaction with Helical Domains ofSaposin C, manuscript. Any lipid known in the art corresponding incharge and length may be used. Samples containing this composition oflipids doped with small amount of saposin C do not destabilize, butlarge aggregates can precipitate out of the solution for the system witha higher concentration of saposin C, indicating destabilization of themembrane. The DOPS/DPPC/DHPC samples are stable over a period of 24months, indicating that the addition of the neutral long chain lipidsand short chain lipids enhance the stability of the aggregates. However,any combination of long and short chain lipids may be used in accordancewith the invention as described herein.

The negative long chain lipids of the present invention may be any longchain phospholipid that has a carbon chain about 14 to about 24 carbonsin length, or about 18 to about 20 carbons in length. An exhaustive listof lipids is available at www.avantilipids.com. One skilled in the artwill appreciate which lipids can be used in the present invention. Whileany combination of long and short chain lipids may be used, somecombinations yield more stable liposomes. For example, while notintending to limit the present invention, the following may guideselection of the composition from which liposomes are formed: wherelong-chains of about 20 to about 24 carbons in length are used,short-chain lipids having lengths of about 6 to about 8 may be used forimproved liposome stability. Where long-chain lengths of about 14 toabout 18 are used, short-chain lipids having lengths of about 6 to about7 may be used for improved liposome stability. While these combinationsof lipids yield more stable liposomes, other combinations maysuccessfully be used, and are not intended to be disclaimed. Table 2illustrates examples of phospholipid combinations that may be used togenerate more stable liposomes. These examples, however, are not meantto imply that other combinations of phospholipids may not be used withthe present invention.

TABLE 2 Table 2. Examples of Combinations of Long and Short-ChainPhosholipids that may be used with the Present Invention, based onPhospholipid Chain Length. The present invention is not limited to thefollowing combinations. Long-Chain Short-Chain Phospholipid LengthPhospholipid Length (Number of Carbons) (Number of Carbons) 14 to 24 4to 8 16 to 22 5 to 7 18 to 20 6 to 7 20 to 24 7 to 8 14 to 18 4 to 6

Further, the presence or absence of saturating hydrocarbons on the lipidchain effect liposome stability. For example, lipids having chainlengths of about 18 or greater are used, the phospholipid may besaturated or unsaturated, preferably unsaturated. For shorter long-chainlipids such as those having about 14 to about 16 carbons, the lipid maybe unsaturated, but use of saturated lipids yields improved performanceof the present invention.

Examples of appropriate lipid ratios are as follows. The molar ratio ofthe selected neutral phospholipid to the selected negative phospholipidin the composition is about 1 to 10 (about 10% neutral phospholipids),or about 1 to 5 (about 20% neutral phospholipids), or about 1 to 1 (50%neutral phospholipids). The molar ratio of the selected long-chainphospholipid to the selected short-chain lipid in the composition isabout 4 to 1 (about 20% short-chain), and can be about 10 to 1 (10%short-chain) to about 3 to 1 (about 33% short-chain). One example of thelong-chain to short chain ratio in one embodiment is as follows:[neutral long-chain lipid]+[acidic long-chain lipid])/[neutralshort-chain lipid] is about 4. As another example, in one embodiment,the molar ratio of DOPS to DPPC in the mixture ranges from about 10-8 to1, or about 7-6 to 1, or about 5-3 to 1 or about 1-2 to 1, with([DPPC]+[DOPS])/DHPC=about 4. Appropriate lipids for use in the presentinvention may be selected from any lipids known in the art or asprovided at www.avantilipids.com.

TABLE 3 Table 3. Hydrodynamic radii (nm) from DLS data of DOPS/DPPC/DHPC aggregates in solution, where [DOPS] + [DPPC])/DHPC = 4. Only thesample with DOPS/DPPC = 10 shows a bimodal distribution. DOPS/DPPCDuration R_(H) nm (%) Ratio (Day) 1-100 100-200 400-800 1 1 40 (79) 145(12) 441 (9)  1 40 42 (76) 173 (7)  705 (17) 5 1 29 (78) 157 (11) 570(11) 5 40 None 147 (51) 689 (49) 10 1 None 138 (70) 582 (30) 10 40 None178 (56) 746 (44) 10 240 None 161 (49) 452 (51) 10 365 None 159 (51) 471(49)

In order for many drugs to have therapeutic potential, it is necessaryfor them to be delivered to the proper location in the body, and thedrugs must have the capability to access the necessary tissues.Liposomes can form the basis for sustained drug release and delivery tospecific cell types, or parts of the body. The therapeutic use ofliposomes also includes the delivery of drugs which are normally toxicin the free form. In the liposomal form, the toxic drug is occluded, andmay be directed away from the tissues sensitive to that drug andtargeted to selected areas. Liposomes can also be used therapeuticallyto release drugs over a prolonged period of time, reducing the frequencyof administration. In addition, liposomes can also provide a method forforming aqueous dispersions of hydrophobic or amphiphilic drugs, whichare normally unsuitable for intravenous delivery.

The liposomes of the present invention may comprise one or morepharmaceutical agent and/or imaging agent that have been trapped in theaqueous interior or between bilayers, or by trapping hydrophobicmolecules within the bilayer. Several techniques can be employed to useliposomes to target encapsulated drugs to selected host tissues, andaway from sensitive tissues. These techniques include manipulating thesize of the liposomes, their net surface charge, and their route ofadministration.

The liposomes of the present invention may also be delivered by apassive delivery route. Passive delivery of liposomes involves the useof various routes of administration, e.g., intravenous, subcutaneous,intramuscular and topical. Each route produces differences inlocalization of the liposomes.

The liposomes of the present invention are also ideal for delivery oftherapeutic or imaging agents across the blood-brain barrier. Thepresent invention relates to a method by which liposomes containingtherapeutic agents can be used to deliver these agents to the CNSwherein the agent is contained within a liposome comprised of the abovereferenced lipids and saposin C, prosaposin or a variant of saposin. Theliposome containing a therapeutic agent can be administered via IVinjection, IM injection, trans-nasal delivery, or any othertransvascular drug delivery method, using generally accepted methods inthe art.

Without intending to be limited by theory, one possible mechanism as tohow saposin-mediated membrane fusion occurs is through proteinconformational changes. Of the pro-saposin derived proteins, saposin Aand saposin C show the highest degree of amino acid identity/similarity.Computationally, both proteins are predicted to fold into amphipathichelical bundle motifs. In general, the saposin-fold is a common supersecondary structure with five amphipathic α-helices folded into a singleglobular domain and is common to both proteins. In one embodiment, thefolding is along a centrally located helix at amino-terminal, againstwhich helices 2 and 3 are packed from one side and helices 4 and 5 fromthe other side. This fold may provide an interface for membraneinteraction.

A mechanism for saposin-mediated membrane fusion with anionicphospholipid membranes is thought to be a two-step process. In the firststep, electrostatic interactions between the positively charged aminoacids (basic form), lysine (Lys) and arginine (Arg), of the saposins andthe negatively charged phospholipid membrane results in an associationbetween these two species (see FIG. 1). In the second step,intramolecular hydrophobic interactions between the helices of twoadjacent saposin proteins brings the two membranes in close enoughproximity for fusion of the membranes to take place (see FIG. 2).

Thus, in accordance with the present invention, the association ofsaposins, and in particular saposin C, with a lipid generally requires apH range from about 5.5 or less since the initial association of saposinC with the membrane arises through an electrostatic interaction of thepositively charged basic amino acid residues of saposin C with theanionic membrane. Thus, it is highly desirable to have the basic aminoacids exist in their protonated forms in order to achieve a high numberof electrostatic interactions.

Alternatively, related fusion proteins and peptides derived from thesaposin family of proteins may not have this lower pH range limitationand thus the pH range of other membrane fusion proteins and peptides canrange from physiological pH (pH of about 7) to lower pH ranges.

Bioactive Agents

In accordance with the present invention, bioactive agents, e.g.,pharmaceutical agents, are contained within the anionic phospholipidmembrane or liposome for saposin-mediated transport within and/orbeneath the dermal and mucosal membranes or across the blood-brainbarrier or other cellular membranes. The active agents may be largebiomolecules including, but not limited to lipids, in particularceramides, steroids, fatty acids, triacylglycerols, genes and proteins,DNA, RNA or siRNA. The active agent may also be comprised of smallorganic molecules. As used herein, “pharmaceutical agent” means anymaterial or mixture of materials which provides a cosmetic ortherapeutic benefit when delivered via saposin C liposomes.

Exemplary bioactive agents or drugs that may be delivered by the systemof the present invention may include, but are not limited to,analgesics, anesthetics, antifungals, antibiotics, anti-inflammatories,anthelmintics, antidotes, antiemetics, antihistamines,antihypertensives, antimalarials, antimicrobials, antipsychotics,antipyretics, antiseptics, antiarthritics, antituberculotics,antitussives, antivirals, cardioactive drugs, cathartics,chemotherapeutic agents, corticoids (steroids), antidepressants,depressants, diagnostic aids, diuretics, enzymes, expectorants,hormones, hypnotics, minerals, nutritional supplements,parasympathomimetics, potassium supplements, sedatives, sulfonamides,stimulants, sympathomimetics, tranquilizers, urinary antiinfectives,vasoconstrictors, vasodilators, vitamins, xanthine derivatives, and thelike.

Digitalis Drugs

Preferred examples of pharmaceutical agents include the digitalis drugs,such as digoxin, digitoxin, digoxigenin, and digitoxigenin. These drugsare all primarily used as cardiac agents.

Steroidal Compounds

Steroidal compounds form another preferred class of pharmaceuticalagent. An example of a steroidal pharmaceutical agent is testosterone(17 beta-hydroxyandrost-4-en-3-one), the principal male steroid. Itsmain therapeutic use is in the treatment of deficient endocrine functionof the testes. Estradiol (estra-1,3,5(10)-triene-3,17 beta-diol) is alsoa preferred steroidal pharmaceutical agent. Estradiol and its esterderivatives are indicated for the treatment of symptoms of menopause andother conditions that cause a deficiency of endogenous estrogenproduction. Progesterone is also a preferred steroidal pharmaceuticalagent. Progesterone is used primarily to suppress or synchronize estrusas well as to control habitual abortion and diagnose and treat menstrualdisorders. Additional preferred steroidal pharmaceutical agents include3-hydroxy-5 alpha-pregnan-20-one, 3-beta-hydroxy-pregn-5-ene-20-one andrelated compounds.

Nonsteroidal Anti-Inflammatory Drugs (NSAID's)

Examples of NSAID's includes piroxicam(4-hydroxy-2-methyl-N-2-pyridinyl-2H-1,2-benzothiazine-3-carboxamide1,1-dioxide), diclofenac, ibuprofen, ketoprofen, meperidine,propoxyphene, nalbuphine, pentazocine, buprenorphine, aspirin,indomethacin, diflunisal, acetaminophen, naproxen, fenoprofen,piroxicam, sulindac, tolmetin, meclofenamate, zomepirac, penicillamine,phenylbutazone, oxyphenbutazone, chloroquine, hydroxychloroquine,azathiaprine, cyclophosphamide, levamisole, prednisone, prednisolone,betamethasone, triamcinolone, and methylprednisolone and indomethacin(1-(4-chlorobenzoyl)-5-methoxy-2-methyl-1H-indole-3-acetic acid).

Amino Acid-Based Drugs

Protein and peptide-based drugs, as well as other amino acid-baseddrugs, may also be used as pharmaceutical agents according to thepresent invention. The problems associated with conventional deliverystrategies for protein and peptide drugs are widely appreciated. Oraladministration of these drugs is generally impractical due todegradation and non-absorption in the gastrointestinal tract. Thus, theparenteral route remains the principal delivery route.

Amino acid-based drugs, such as the cephalosporins, will typically havemolecular weight less than about 5000, and preferably, less than about2500, and more preferably, less than about 1000. Protein and peptidedrugs typically have a molecular weight of at least about 100 daltons,and more typically a molecular weight in the range of about 200 to40,000 daltons. Examples of peptides and proteins in this size rangeinclude, but are not limited to luteinizing hormone-releasing hormone,somatostatin, bradykinin, goserelin, somatotropin, buserelin,platelet-derived growth factor, triptorelin, gonadorelin, asparaginase,nafarelin, bleomycin sulfate, leuprolide, chymopapain, growthhormone-releasing factor, parathyroid hormone (PTH), cholecystokinin,chorionic gonadotropin, insulin, corticotropin (ACTH), calcitoninerythropoietin, glucagon, hyaluronidase, interferons, e.g., alpha,interleukins, e.g., IL-1 thyrotropin-releasing hormone, menotropins,pituitary hormones (e.g., urofollitropin (Follicle hGH, hMG, hCG, FSH,etc.), melanocyte-stimulating hormone, gonadotropin releasing hormone,oxytocin, vasopressin, streptokinase, tissue plasminogen activator,angiotensin ii antagonists, bradykinin potentiatorB, bradykininantagonists, bradykinin potentiator C, enkephalins, insulin-like growthfactors, prostaglandin antagonists, tumor necrosis factor, epidermalgrowth factor (ego, amylin, lipotropin, and thyroid stimulating hormone.

An example of a preferred peptide pharmaceutical agent is parathyroidhormone (PTH) (see Harper et al., Eds., Review of PhysiologicalChemistry, 16th Ed., Lange Medical Publications, Los Altos, Calif.(1977) p. 468). Also, a fragment consisting of about 34 amino acidresidues from the N-terminal has been isolated and found to display thefull biological activity of PTH (see Potts et al., in ParathyroidHormone and Thyrocalcitonin (Calcitonin), R. V. Talmage, et al., Eds.Excerpta Medica, New York (1968)). The sequence of the polypeptidevaries slightly among mammalian species. According to the presentinvention, PTH is meant to include human parathyroid hormone, as well asthe other variants and the 34 amino acid fragment. PTH serves as aregulatory factor in the homeostatic control of calcium and phosphatemetabolism (see, e.g., Parsons, et al. “Physiology and Chemistry ofParathyroid Hormone” in Clinics in Endocrinology and Metabolism, I.MacIntyre, Ed. Saunders, Philadelphia (1972) pp. 33-78). The maintherapeutic use for PTH is in the treatment of osteoporosis. PTH hasalso been used as a blood calcium regulator.

In one embodiment, calcitonin is also a preferred peptide pharmaceuticalagent. Calcitonin is a polypeptide containing 32 amino acid residues(see Harper et al., Eds., Review of Physiological Chemistry, 16th Ed.,Lange Medical Publications, Los Altos, Calif. (1977), p. 469). Accordingto the present invention, calcitonin is meant to include all calcitonin,including that of humans, mammals, and fish, as well as other variants.Calcitonin is a calcium regulating hormone and has been used in thetreatment of osteoporosis, hypercalcemia, and Paget's disease.

An additional preferred protein drug is the cytokine IL-10. IL-10 isproduced by the TH2 helper subset, B cell subsets and LPs-activatedmonocytes. IL-10 inhibits several immune functions that are relevant tothe skin immune response and thus, the development of the irritation andinflammation that is sometimes associated with the transdermal deliveryof drugs. More specifically, the release of IFN-alpha, which initiatesthe cascade of cellular activation leading to the skin's immuneresponse, is inhibited by IL-10. IL-10 also suppresses the synthesis ofnumerous pro-inflammatory cytokines by macrophages, as well as theproliferation of antigen-specific T cell proliferation by downregulating class II MHC expression.

Nucleic Acid-Based Drugs

Generally, nucleic acid-based drugs have had limited success astherapeutic agents, in part, because of problems associated with theirstability and delivery. Nucleotide-based pharmaceutical agentsfrequently contain a phosphodiester bond which is sensitive todegradation by nucleases. Such degradation would be a significantimpediment to the use of an oligonucleotide or nucleic acid as apharmaceutical agent that depends upon the integrity of the sequence forits recognition specificity. Thus, naturally occurring oligonucleotidesand nucleic acids often must typically be chemically modified to renderthem resistant to nucleases which would degrade them in vivo, or even invitro unless care is taken to choose appropriate conditions. However,this is not necessary using the drug delivery system of the presentinvention.

The nucleotide-based drugs of the present invention include aptamers,antisense compounds, and triple helix drugs. The nucleotide-based drugstypically will have a molecular weight greater than about 350 and mayrange up to about 100 bases. Examples of nucleotide-based drugs includedi- and trinucleotides, such as GS 375, a dinucleotide analog withpotential therapeutic activity against the influenza virus (GileadSciences, Inc., Foster City, Calif.).

In one embodiment, the nucleotide-based drug comprises one or moretherapeutic genes. The therapeutic gene which is encapsulated within theliposome can be any of the common therapeutic genes which are used toexpress therapeutic and diagnostic agents. Exemplary therapeutic genesinclude brain-derived neurotrophic factor (BDNF) for treatment ofneurodegenerative disease, stroke, or brain trauma; tyrosine hydroxylaseand/or aromatic amino acid decarboxylase for Parkinson's disease;.beta.-glucuronidase; hexosaminidase A; herpes simplex virus thymidinekinase or genes encoding antisense RNA to the epidemal growth factorreceptor for treatment of brain tumors; lysosomal storage disorderreplacement enzymes for Tay-Sachs and other lysosomal storage disorders;gene encoding antisense RNA for the treatment of the cerebral componentof acquired immune deficiency syndrome (AIDS). In addition to thetherapeutic gene, the plasmid DNA may also contain DNA sequences eitherbefore or after the therapeutic sequence and these additional parts ofthe plasmid may promote tissue-specific transcription of the plasmid ina particular cell in the brain, may promote enhanced translation and/orstabilization of the mRNA of the therapeutic gene, and may enableepisomal replication of the transgene in brain cells. In general, thetherapeutic gene will contain at least 100 nucleotides or have amolecular weight above 30,000 Daltons. It is preferred that thetherapeutic gene be contained within a plasmid or other suitable carrierfor encapsulation within the internal compartment of the liposome ornanocontainer.

A therapeutic gene may be encapsulated within the liposome according toany of the well known drug encapsulation processes. For example,encapsulation by sonication, freeze/thaw, evaporation, and extrusionthrough membrane filters.

The number of therapeutic genes encapsulated within the liposome mayvary from 1 to many, depending on the disease being treated. Thelimiting factor will be the diameter of therapeutic gene that isencapsulated within the liposome. Using polycationic proteins such ashistone, protamine, or polylysine, it is possible to compact the size ofplasmid DNA that contains several thousand nucleotides to a structurethat has a diameter of 10-30 nm. The volume of a 100 diameter liposomeis 1000-fold and 35-fold greater than the volume of a 10 nm and 30 nmDNA compacted sphere, respectively. Therefore, it is possible toencapsulate many copies of the same gene or multiple copies of multiplegenes within the liposome.

Bioactive agents include oligomers such as (1) antisense compounds and(2) other bioactive oligomers. As used herein, the term “antisensecompound” encompasses, inter alia, single stranded antisenseoligonucleotides (DNA, DNA-like, RNA, RNA-like) or double stranded orself-hybridizing constructs comprising an antisense orientationoligonucleotide, antisense PNAs, ribozymes and EGSs (described infra).Antisense compounds can exert their effect by a variety of means. Onesuch means is the antisense-mediated direction of an endogenousnuclease, such as RNase H in eukaryotes or RNase P in prokaryotes, ordsRNAases in RNAi pathways to the target nucleic acid (Chiang et al., J.Biol. Chem., 1991, 266, 18162; Forster et al., Science, 1990, 249, 783).The sequences that recruit RNase P are known as External GuideSequences, hence the abbreviation “EGSs” (Guerrier-Takada et al., Proc.Natl. Acad. Sci. USA, 1997, 94, 8468).

Another type of bioactive oligomer is an RNA-RNA hybrid molecule thatcan modulate gene expression. The double strand RNA may in someinstances be described as siRNA. For the purposes of describing anembodiment of this invention, an siRNA is a combination of an antisensestrand and a sense strand, each of a specified length sufficient toexhibit desirable properties such as a stability and target specificity,for example from about 8-30, about 12-27, about 17-25, or about 19-23nucleotides long. Such a complementary pair of oligonucleotides can beblunt ended or can include additional nucleotides on either or both oftheir 5′ or 3′ ends. Further they can include other molecules ormolecular structures on their 3′ or 5′ ends such as a phosphate group onthe 5′ end. A preferred group of compounds of the invention include aphosphate group on the 5′ end of the antisense strand compound. Otherpreferred compounds also include a phosphate group on the 5′ end of thesense strand compound. An even further preferred compounds would includeadditional nucleotides such as a two base overhang on the 3′ end.

The term “other bioactive oligomer” encompasses, inter alia, aptamersand molecular decoys. As used herein, the term is meant to refer to anyoligonucleotide (including an RNA or PNA) that (1) provides aprophylactic, palliative or therapeutic effect to an animal in needthereof and (2) acts by a non-antisense mechanism, i.e., by some meansother than by hybridizing to a nucleic acid.

In one embodiment, the bioactive agent is an aptamer or molecular decoy.Aptamers are single-stranded oligonucleotides that bind specific ligandsvia a mechanism other than Watson-Crick base pairing. Aptamers aretypically targeted to, e.g., a protein and are not designed to bind to anucleic acid (Ellington et al., Nature, 1990, 346, 818).

Molecular decoys are short double-stranded nucleic acids (includingsingle-stranded nucleic acids designed to “fold back” on themselves)that mimic a site on a nucleic acid to which a factor, such as aprotein, binds. Such decoys are expected to competitively inhibit thefactor; that is, because the factor molecules are bound to an excess ofthe decoy, the concentration of factor bound to the cellular sitecorresponding to the decoy decreases, with resulting therapeutic,palliative or prophylactic effects. Methods of identifying andconstructing decoy molecules are described in, e.g., U.S. Pat. No.5,716,780 to Edwards et al.

Another type of bioactive oligomer is an RNA-DNA hybrid molecule thatcan direct gene conversion of an endogenous nucleic acid (Cole-Strausset al., Science, 1996, 273, 1386). Any of the preceding bioactiveoligomers may be formulated in the liposomes of the invention and usedfor prophylactic or therapeutic purposes.

Some embodiments of the invention, a single oligonucleotide having boththe antisense portion as a first region in the oligonucleotide and thesense portion as a second region in the oligonucleotide is selected. Thefirst and second regions are linked together by either a nucleotidelinker (a string of one or more nucleotides that are linked together ina sequence) or by a non-nucleotide linker region or by a combination ofboth a nucleotide and non-nucleotide structure. In each of thesestructures, the oligonucleotide, when folded back on itself, would becomplementary at least between the first region, the antisense portion,and the second region, the sense portion. Thus the oligonucleotide wouldhave a palindrome within it structure wherein the first region, theantisense portion in the 5′ to 3′ direction, is complementary to thesecond region, the sense portion in the 3′ to 5′ direction.

In further embodiments, the invention includes anoligonucleotide/protein composition. This composition has both anoligonucleotide component and a protein component. The oligonucleotidecomponent comprises at least one oligonucleotide, either the antisenseor the sense oligonucleotide but preferable the antisenseoligonucleotide (the oligonucleotide that is antisense to the targetnucleic acid). The protein component of the composition comprises atleast one protein that forms a portion of the RNA-induced silencingcomplex, i.e., the RISC complex. The oligonucleotide component can alsocomprise both the antisense and the sense strand oligonucleotides.

RISC is a ribonucleoprotein complex that contains an oligonucleotidecomponent and proteins of the Argonaute family of proteins. While we donot wish to be bound by theory, the Argonaute proteins are a class ofproteins, some of which have been shown to contain a PAZ and Piwi domainand that have been implicated in processes previously linked toposttranscriptional silencing. The Argonaute family of proteinsincludes, but depending on species, are not necessary limited to e1F2C1and e1F2C2. e1F2C2 is also known as human GERp95. While we do not wishto be bound by theory, at least the antisense oligonucleotide strand isbound to the protein component to form the RISC complex. Additional, thecomplex might also include the sense strand oligonucleotide.

The oligomeric compounds of the invention may be used in the form ofsingle-stranded, double-stranded, circular or hairpin oligomericcompounds and may contain structural elements such as internal orterminal bulges or loops. Once introduced to a system, the oligomericcompounds of the invention may elicit the action of one or more enzymesor proteins to effect modification of the target nucleic acid.

One non-limiting example of such a protein is the RISC complex. Use ofthe RISC complex to effect cleavage of RNA targets thereby greatlyenhances the efficiency of oligonucleotide-mediated inhibition of geneexpression. Similar roles have been postulated for other ribonucleasessuch as those in the RNase III and ribonuclease L family of enzymes.

In another embodiment, the oligomeric compound of the invention includea single-stranded antisense oligonucleotide that binds in a RISCcomplex, a double stranded antisense/sense pair of oligonucleotide or asingle strand oligonucleotide that includes both an antisense portionand a sense portion. Each of these compounds or compositions is used toinduce potent and specific modulation of gene function. Such specificmodulation of gene function has been shown in many species by theintroduction of double-stranded structures, such as double-stranded RNA(dsRNA) molecules and has been shown to induce potent and specificantisense-mediated reduction of the function of a gene or its associatedgene products. This phenomenon occurs in both plants and animals and isbelieved to have an evolutionary connection to viral defense andtransposon silencing.

Aptamers (or nucleic acid antibody) are single- or double-stranded DNAor single-stranded RNA molecules that bind specific molecular targets.Generally, aptamers function by inhibiting the actions of the moleculartarget, e.g., proteins, by binding to the pool of the target circulatingin the blood. Examples of aptamers include Gilead's antithrombininhibitor GS 522 and its derivatives (Gilead Science, Foster City,Calif.; see also Macaya et al. (1993) Proc. Natl. Acad. Sci. USA90:3745-9; Bock et al. (1992) Nature (London) 355:564-566; and Wang etal. (1993) Biochem. 32:1899-904). Similarly, siRNA (small interferingRNA molecules) as known in the art may be used with the presentinvention. See FIGS. 8 and 9.

For diseases that result from the inappropriate expression of genes,specific prevention or reduction of the expression of such genesrepresents an ideal therapy. In principle, production of a particulargene product may be inhibited, reduced or shut off by hybridization of asingle-stranded deoxynucleotide or ribodeoxynucleotide complementary toan accessible sequence in the mRNA, or a sequence within the transcriptwhich is essential for pre-mRNA processing, or to a sequence within thegene itself. This paradigm for genetic control is often referred to asantisense or antigene inhibition.

Antisense compounds are oligonucleotides that are designed to bind anddisable or prevent the production of the mRNA responsible for generatinga particular protein. Antisense compounds can provide a therapeuticfunction by inhibiting in vivo the formation of one or more proteinsthat cause or are involved with disease. Antisense compoundscomplementary to certain gene messenger RNA or viral sequences have beenreported to inhibit the spread of disease related to viral andretroviral infectious agents (see, for example, Matsukura et al. (1987)Proc. Natl. Acad. Sci. USA 84:7706, and references cited therein).Others have reported that oligonucleotides can bind to duplex DNA viatriple helix formation and inhibit transcription and/or DNA synthesis.

Antisense compounds include antisense RNA or DNA, single or doublestranded, oligonucleotides, or their analogs, which can hybridizespecifically to individual mRNA species and prevent transcription and/orRNA processing of the mRNA species and/or translation of the encodedpolypeptide and thereby effect a reduction in the amount of therespective encoded polypeptide (see Ching et al. Proc. Natl. Acad. Sci.U.S.A. 86:10006-10010 (1989); Broder et al. Ann. Int. Med. 113:604-618(1990); Loreau et al. FEBS Letters 274:53-56 (1990)).

Triple helix compounds (also referred to as triple strand drugs) areoligonucleotides that bind to sequences of double-stranded DNA and areintended to inhibit selectively the transcription of disease-causinggenes, such as viral genes, e.g., HIV and herpes simplex virus, andoncogenes, i.e., they stop protein production at the cell nucleus. Thesedrugs bind directly to the double stranded DNA in the cell's genome toform a triple helix and thus, prevents the cell from making a targetprotein (see, for example U.S. Pat. No. 5,176,996, Hogan et al, Jan. 5,1993).

The site specificity of oligonucleotides (e.g., antisense compounds andtriple helix drugs) is not significantly affected by modification of thephosphodiester linkage or by chemical modification of theoligonucleotide terminus. Consequently, these oligonucleotides can bechemically modified; enhancing the overall binding stability, increasingthe stability with respect to chemical degradation, increasing the rateat which the oligonucleotides are transported into cells, and conferringchemical reactivity to the molecules. The general approach toconstructing various oligonucleotides useful in antisense therapy hasbeen reviewed by vander Krol et al. (1988) Biotechniques 6:958-976 andStein et al. (1988) Cancer Res. 48:2659-2668.

Accordingly, aptamers, antisense compounds and triple helix drugs alsocan include nucleotide substitutions, additions, deletions, ortranspositions, so long as specific hybridization to or association withthe relevant target sequence is retained as a functional property of theoligonucleotide. For example, some embodiments will employphosphorothioate analogs which are more resistant to degradation bynucleases than their naturally occurring phosphate diester counterpartsand are thus expected to have a higher persistence in vivo and greaterpotency (see, Campbell et al. (1990) J. Biochem. Biophys. Methods20:259-267). Phosphoramidate derivatives of oligonucleotides also areknown to bind to complementary polynucleotides and have the additionalcapability of accommodating covalently attached ligand species and willbe amenable to the methods of the present invention (see Froehler et al.(1988) Nucleic Acids Res. 16(11): 4831).

In addition, nucleotide analogs, for example where the sugar or base ischemically modified, can be employed in the present invention. Analogousforms of purines and pyrimidines are those generally known in the art,many of which are used as chemotherapeutic agents.

Terminal modification also provides a useful procedure to modify celltype specificity, pharmacokinetics, nuclear permeability, and absolutecell uptake rate for oligonucleotide pharmaceutical agents. For example,substitutions at the 5′ and 3′ ends include reactive groups which allowcovalent crosslinking of the nucleotide-based pharmaceutical agent toother species and bulky groups which improve cellular uptake (seeOligodeoxynucleotides: Antisense Inhibitors of Gene Expression, (1989)Cohen, Ed., CRC Press; Prospects for Antisense Nucleic Acid Therapeuticsfor Cancer and AIDS, (1991), Wickstrom, Ed., Wiley-Liss; GeneRegulation: Biology of Antisense RNA and DNA, (1992) Erickson and Izant,Eds., Raven Press; and Antisense RNA and DNA, (1992), Murray, Ed.,Wiley-Liss. For general methods relating to antisense compounds, seeAntisense RNA and DNA, (1988), D. A. Melton, Ed., Cold Spring HarborLaboratory, Cold Spring Harbor, N.Y.).

A polynucleotide can be delivered to a cell to express an exogenousnucleotide sequence, to inhibit, eliminate, augment, or alter expressionof an endogenous nucleotide sequence, or to affect a specificphysiological characteristic not naturally associated with the cell. Thepolynucleotide can be a sequence whose presence or expression in a cellalters the expression or function of cellular genes or RNA. A deliveredpolynucleotide can stay within the cytoplasm or nucleus apart from theendogenous genetic material. Alternatively, DNA can recombine with(become a part of) the endogenous genetic material. Recombination cancause DNA to be inserted into chromosomal DNA by either homologous ornon-homologous recombination.

A polynucleotide-based gene expression inhibitor comprises anypolynucleotide containing a sequence whose presence or expression in acell causes the degradation of or inhibits the function, transcription,or translation of a gene in a sequence-specific manner.Polynucleotide-based expression inhibitors may be selected from thegroup comprising: siRNA, microRNA, interfering RNA or RNAi, dsRNA,ribozymes, antisense polynucleotides, and DNA expression cassettesencoding siRNA, microRNA, dsRNA, ribozymes or antisense nucleic acids.SiRNA comprises a double stranded structure typically containing 15-50base pairs and preferably 19-25 base pairs and having a nucleotidesequence identical or nearly identical to an expressed target gene orRNA within the cell. An siRNA may be composed of two annealedpolynucleotides or a single polynucleotide that forms a hairpinstructure. MicroRNAs (miRNAs) are small noncoding polynucleotides, about22 nucleotides long, that direct destruction or translational repressionof their mRNA targets. Antisense polynucleotides comprise sequence thatis complimentary to a gene or mRNA. Antisense polynucleotides include,but are not limited to: morpholinos, 2′-O-methyl polynucleotides, DNA,RNA and the like. The polynucleotide-based expression inhibitor may bepolymerized in vitro, recombinant, contain chimeric sequences, orderivatives of these groups. The polynucleotide-based expressioninhibitor may contain ribonucleotides, deoxyribonucleotides, syntheticnucleotides, or any suitable combination such that the target RNA and/orgene is inhibited.

Polynucleotides may contain an expression cassette coded to express awhole or partial protein, or RNA. An expression cassette refers to anatural or recombinantly produced polynucleotide that is capable ofexpressing a sequence. The cassette contains the coding region of thegene of interest along with any other sequences that affect expressionof the sequence of interest. An expression cassette typically includes apromoter (allowing transcription initiation), and a transcribedsequence. Optionally, the expression cassette may include, but is notlimited to, transcriptional enhancers, non-coding sequences, splicingsignals, transcription termination signals, and polyadenylation signals.An RNA expression cassette typically includes a translation initiationcodon (allowing translation initiation), and a sequence encoding one ormore proteins. Optionally, the expression cassette may include, but isnot limited to, translation termination signals, a polyadenosinesequence, internal ribosome entry sites (IRES), and non-codingsequences. The polynucleotide may contain sequences that do not serve aspecific function in the target cell but are used in the generation ofthe polynucleotide. Such sequences include, but are not limited to,sequences required for replication or selection of the polynucleotide ina host organism.

In certain embodiments of the invention, as noted above, saposinfusogenic membranes or liposomes are used to facilitate delivery oflarger nucleic acid molecules than conventional siNAs, including largenucleic acid precursors of siNAs. For example, the methods andcompositions herein may be employed for enhancing delivery of largernucleic acids that represent “precursors” to desired siNAs, wherein theprecursor amino acids may be cleaved or otherwise processed before,during or after delivery to a target cell to form an active siNA formodulating gene expression within the target cell. For example, a siNAprecursor polynucleotide may be selected as a circular, single-strandedpolynucleotide, having two or more loop structures and a stem comprisingself-complementary sense and antisense regions, wherein the antisenseregion comprises a nucleotide sequence that is complementary to anucleotide sequence in a target nucleic acid molecule or a portionthereof, and the sense region having nucleotide sequence correspondingto the target nucleic acid sequence or a portion thereof, and whereinthe circular polynucleotide can be processed either in vivo or in vitroto generate an active siNA molecule capable of mediating RNAi.

In mammalian cells, dsRNAs longer than 30 base pairs can activate thedsRNA-dependent kinase PKR and 2′-5′-oligoadenylate synthetase, normallyinduced by interferon. The activated PKR inhibits general translation byphosphorylation of the translation factor eukaryotic initiation factor2.alpha.(eIF2.alpha.), while 2′-5′-oligoadenylate synthetase causesnonspecific mRNA degradation via activation of RNase L. By virtue oftheir small size (referring particularly to non-precursor forms),usually less than 30 base pairs, and most commonly between about 17-19,19-21, or 21-23 base pairs, the siNAs of the present invention avoidactivation of the interferon response.

In contrast to the nonspecific effect of long dsRNA, siRNA can mediateselective gene silencing in the mammalian system. Hairpin RNAs, with ashort loop and 19 to 27 base pairs in the stem, also selectively silenceexpression of genes that are homologous to the sequence in thedouble-stranded stem. Mammalian cells can convert short hairpin RNA intosiRNA to mediate selective gene silencing.

RISC mediates cleavage of single stranded RNA having sequencecomplementary to the antisense strand of the siRNA duplex. Cleavage ofthe target RNA takes place in the middle of the region complementary tothe antisense strand of the siRNA duplex. Studies have shown that 21nucleotide siRNA duplexes are most active when containing two nucleotide3′-overhangs. Furthermore, complete substitution of one or both siRNAstrands with 2′-deoxy (2′-H) or 2T-O-methyl nucleotides abolishes RNAiactivity, whereas substitution of the 3′-terminal siRNA overhangnucleotides with deoxy nucleotides (2′-H) has been reported to betolerated.

Studies have shown that replacing the 3′-overhanging segments of a21-mer siRNA duplex having 2 nucleotide 3′ overhangs withdeoxyribonucleotides does not have an adverse effect on RNAi activity.Replacing up to 4 nucleotides on each end of the siRNA withdeoxyribonucleotides has been reported to be well tolerated whereascomplete substitution with deoxyribonucleotides results in no RNAiactivity.

Alternatively, the siNAs can be delivered as single or multipletranscription products expressed by a polynucleotide vector encoding thesingle or multiple siNAs and directing their expression within targetcells. In these embodiments the double-stranded portion of a finaltranscription product of the siRNAs to be expressed within the targetcell can be, for example, 15 to 49 bp, 15 to 35 bp, or about 21 to 30 bplong. Within exemplary embodiments, double-stranded portions of siNAs,in which two strands pair up, are not limited to completely pairednucleotide segments, and may contain nonpairing portions due to mismatch(the corresponding nucleotides are not complementary), bulge (lacking inthe corresponding complementary nucleotide on one strand), overhang, andthe like. Nonpairing portions can be contained to the extent that theydo not interfere with siNA formation. In more detailed embodiments, a“bulge” may comprise 1 to 2 nonpairing nucleotides, and thedouble-stranded region of siNAs in which two strands pair up may containfrom about 1 to 7, or about 1 to 5 bulges. In addition, “mismatch”portions contained in the double-stranded region of siNAs may be presentin numbers from about 1 to 7, or about 1 to 5. Most often in the case ofmismatches, one of the nucleotides is guanine, and the other is uracil.Such mismatching may be attributable, for example, to a mutation from Cto T, G to A, or mixtures thereof, in a corresponding DNA coding forsense RNA, but other cause are also contemplated. Furthermore, in thepresent invention the double-stranded region of siNAs in which twostrands pair up may contain both bulge and mismatched portions in theapproximate numerical ranges specified.

The terminal structure of siNAs of the invention may be either blunt orcohesive (overhanging) as long as the siNA retains its activity tosilence expression of target genes. The cohesive (overhanging) endstructure is not limited only to the 3′ overhang as reported by others.On the contrary, the 5′ overhanging structure may be included as long asit is capable of inducing a gene silencing effect such as by RNAi. Inaddition, the number of overhanging nucleotides is not limited toreported limits of 2 or 3 nucleotides, but can be any number as long asthe overhang does not impair gene silencing activity of the siNA. Forexample, overhangs may comprise from about 1 to 8 nucleotides, moreoften from about 2 to 4 nucleotides. The total length of siNAs havingcohesive end structure is expressed as the sum of the length of thepaired double-stranded portion and that of a pair comprising overhangingsingle-strands at both ends. For example, in the exemplary case of a 19bp double-stranded RNA with 4 nucleotide overhangs at both ends, thetotal length is expressed as 23 bp. Furthermore, since the overhangingsequence may have low specificity to a target gene, it is notnecessarily complementary (antisense) or identical (sense) to the targetgene sequence. Furthermore, as long as the siNA is able to maintain itsgene silencing effect on the target gene, it may contain low molecularweight structure (for example a natural RNA molecule such as tRNA, rRNAor viral RNA, or an artificial RNA molecule), for example, in theoverhanging portion at one end.

In addition, the terminal structure of the siNAs may have a stem-loopstructure in which ends of one side of the double-stranded nucleic acidare connected by a linker nucleic acid, e.g., a linker RNA. The lengthof the double-stranded region (stem-loop portion) can be, for example,15 to 49 bp, often 15 to 35 bp, and more commonly about 21 to 30 bplong. Alternatively, the length of the double-stranded region that is afinal transcription product of siNAs to be expressed in a target cellmay be, for example, approximately 15 to 49 bp, 15 to 35 bp, or about 21to 30 bp long. When linker segments are employed, there is no particularlimitation in the length of the linker as long as it does not hinderpairing of the stem portion. For example, for stable pairing of the stemportion and suppression of recombination between DNAs coding for thisportion, the linker portion may have a clover-leaf tRNA structure. Evenif the linker has a length that would hinder pairing of the stemportion, it is possible, for example, to construct the linker portion toinclude introns so that the introns are excised during processing of aprecursor RNA into mature RNA, thereby allowing pairing of the stemportion. In the case of a stem-loop siRNA, either end (head or tail) ofRNA with no loop structure may have a low molecular weight RNA. Asdescribed above, these low molecular weight RNAs may include a naturalRNA molecule, such as tRNA, rRNA or viral RNA, or an artificial RNAmolecule.

The siNA can also comprise a single stranded polynucleotide havingnucleotide sequence complementary to nucleotide sequence in a targetnucleic acid molecule or a portion thereof (for example, where such siNAmolecule does not require the presence within the siNA molecule ofnucleotide sequence corresponding to the target nucleic acid sequence ora portion thereof), wherein the single stranded polynucleotide canfurther comprise a terminal phosphate group, such as a 5′-phosphate (seefor example Martinez et al., Cell., 110: 563-574 (2002) and Schwarz etal., Molecular Cell, 10: 537-568(2002), or 5′,3′-diphosphate.

As used herein, the term siNA molecule is not limited to moleculescontaining only naturally-occurring RNA or DNA, but also encompasseschemically-modified nucleotides and non-nucleotides. In certainembodiments, the short interfering nucleic acid molecules of theinvention lack T-hydroxy (2′-OH) containing nucleotides. In certainembodiments short interfering nucleic acids do not require the presenceof nucleotides having a 2′-hydroxy group for mediating RNAi and as such,short interfering nucleic acid molecules of the invention optionally donot include any ribonucleotides (e.g., nucleotides having a 2′—OHgroup). Such siNA molecules that do not require the presence ofribonucleotides within the siNA molecule to support RNAi can howeverhave an attached linker or linkers or other attached or associatedgroups, moieties, or chains containing one or more nucleotides with2′—OH groups. Optionally, siNA molecules can comprise ribonucleotides atabout 5, 10, 20, 30, 40, or 50% of the nucleotide positions.

As used herein, the term siNA is meant to be equivalent to other termsused to describe nucleic acid molecules that are capable of mediatingsequence specific RNAi, for example short interfering RNA (siRNA),double-stranded RNA (dsRNA), micro-RNA (mRNA), short hairpin RNA(shRNA), short interfering oligonucleotide, short interfering nucleicacid, short interfering modified oligonucleotide, chemically-modifiedsiRNA, post-transcriptional gene silencing RNA (ptgsRNA), and others.

In other embodiments, siNA molecules for use within the invention maycomprise separate sense and antisense sequences or regions, wherein thesense and antisense regions are covalently linked by nucleotide ornon-nucleotide linker molecules, or are alternately non-covalentlylinked by ionic interactions, hydrogen bonding, van der waalsinteractions, hydrophobic interactions, and/or stacking interactions.

“Antisense RNA” is an RNA strand having a sequence complementary to atarget gene MRNA, and thought to induce RNAi by binding to the targetgene MRNA. “Sense RNA” has a sequence complementary to the antisenseRNA, and annealed to its complementary antisense RNA to form siRNA.These antisense and sense RNAs have been conventionally synthesized withan RNA synthesizer. As used herein, the term “RNAi construct” is ageneric term used throughout the specification to include smallinterfering RNAs (siRNAs), hairpin RNAs, and other RNA species which canbe cleaved in vivo to form siRNAs. RNAi constructs herein also includeexpression vectors (also referred to as RNAi expression vectors) capableof giving rise to transcripts which form dsRNAs or hairpin RNAs incells, and/or transcripts which can produce siRNAs in vivo. Optionally,the siRNA include single strands or double strands of siRNA.

An siHybrid molecule is a double-stranded nucleic acid that has asimilar function to siRNA. Instead of a double-stranded RNA molecule, ansiHybrid is comprised of an RNA strand and a DNA strand. Preferably, theRNA strand is the antisense strand as that is the strand that binds tothe target mRNA. The siHybrid created by the hybridization of the DNAand RNA strands have a hybridized complementary portion and preferablyat least one 3′ overhanging end.

siNAs for use within the invention can be assembled from two separateoligonucleotides, where one strand is the sense strand and the other isthe antisense strand, wherein the antisense and sense strands areself-complementary (i.e. each strand comprises nucleotide sequence thatis complementary to nucleotide sequence in the other strand; such aswhere the antisense strand and sense strand form a duplex or doublestranded structure, for example wherein the double stranded region isabout 19 base pairs). The antisense strand may comprise a nucleotidesequence that is complementary to a nucleotide sequence in a targetnucleic acid molecule or a portion thereof, and the sense strand maycomprise a nucleotide sequence corresponding to the target nucleic acidsequence or a portion thereof Alternatively, the siNA can be assembledfrom a single oligonucleotide, where the self-complementary sense andantisense regions of the siNA are linked by means of a nucleicacid-based or non-nucleic acid-based linker(s).

Within additional embodiments, siNAs for intracellular deliveryaccording to the methods and compositions of the invention can be apolynucleotide with a duplex, asymmetric duplex, hairpin or asymmetrichairpin secondary structure, having self-complementary sense andantisense regions, wherein the antisense region comprises a nucleotidesequence that is complementary to a nucleotide sequence in a separatetarget nucleic acid molecule or a portion thereof, and the sense regioncomprises a nucleotide sequence corresponding to the target nucleic acidsequence or a portion thereof.

Non-limiting examples of chemical modifications that can be made in ansiNA include without limitation phosphorothioate internucleotidelinkages, 2′-deoxyribonucleotides, 2′-O-methyl ribonucleotides,2′-deoxy-2′-fluoro ribonucleotides, “universal base” nucleotides,“acyclic” nucleotides, 5-C-methyl nucleotides, and terminal glyceryland/or inverted deoxy abasic residue incorporation. These chemicalmodifications, when used in various siNA constructs, are shown topreserve RNAi activity in cells while at the same time, dramaticallyincreasing the serum stability of these compounds.

In a non-limiting example, the introduction of chemically-modifiednucleotides into nucleic acid molecules provides a powerful tool inovercoming potential limitations of in vivo stability andbioavailability inherent to native RNA molecules that are deliveredexogenously. For example, the use of chemically-modified nucleic acidmolecules can enable a lower dose of a particular nucleic acid moleculefor a given therapeutic effect since chemically-modified nucleic acidmolecules tend to have a longer half-life in serum. Furthermore, certainchemical modifications can improve the bioavailability of nucleic acidmolecules by targeting particular cells or tissues and/or improvingcellular uptake of the nucleic acid molecule. Therefore, even if theactivity of a chemically-modified nucleic acid molecule is reduced ascompared to a native nucleic acid molecule, for example, when comparedto an all-RNA nucleic acid molecule, the overall activity of themodified nucleic acid molecule can be greater than that of the nativemolecule due to improved stability and/or delivery of the molecule.Unlike native unmodified siNA, chemically-modified siNA can alsominimize the possibility of activating interferon activity in humans.

The siNA molecules described herein, the antisense region of a siNAmolecule of the invention can comprise a phosphorothioateinternucleotide linkage at the 3′-end of said antisense region. In anyof the embodiments of siNA molecules described herein, the antisenseregion can comprise about one to about five phosphorothioateinternucleotide linkages at the 5′-end of said antisense region. In anyof the embodiments of siNA molecules described herein, the 3′-terminalnucleotide overhangs of a siNA molecule of the invention can compriseribonucleotides or deoxyribonucleotides that are chemically-modified ata nucleic acid sugar, base, or backbone. In any of the embodiments ofsiNA molecules described herein, the 3′-terminal nucleotide overhangscan comprise one or more universal base ribonucleotides. In any of theembodiments of siNA molecules described herein, the 3′-terminalnucleotide overhangs can comprise one or more acyclic nucleotides.

For example, in a non-limiting example, the invention features achemically-modified short interfering nucleic acid (siNA) having about1, 2, 3, 4, 5, 6, 7, 8 or more phosphorothioate internucleotide linkagesin one siNA strand. In yet another embodiment, the invention features achemically-modified short interfering nucleic acid (siNA) individuallyhaving about 1, 2, 3, 4, 5, 6, 7, 8 or more phosphorothioateinternucleotide linkages in both siNA strands. The phosphorothioateinternucleotide linkages can be present in one or both oligonucleotidestrands of the siNA duplex, for example in the sense strand, theantisense strand, or both strands. The siNA molecules of the inventioncan comprise one or more phosphorothioate internucleotide linkages atthe 3′-end, the 5′-end, or both of the 3′- and 5′-ends of the sensestrand, the antisense strand, or both strands. For example, an exemplarysiNA molecule of the invention can comprise about 1 to about 5 or more(e.g., about 1, 2, 3, 4, 5, or more) consecutive phosphorothioateinternucleotide linkages at the 5′-end of the sense strand, theantisense strand, or both strands. In another non-limiting example, anexemplary siNA molecule of the invention can comprise one or more (e.g.,about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) pyrimidinephosphorothioate internucleotide linkages in the sense strand, theantisense strand, or both strands. In yet another non-limiting example,an exemplary siNA molecule of the invention can comprise one or more(e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) purinephosphorothioate internucleotide linkages in the sense strand, theantisense strand, or both strands.

An siNA molecule may be comprised of a circular nucleic acid molecule,wherein the siNA is about 38 to about 70 (e.g., about 38, 40, 45, 50,55, 60, 65, or 70) nucleotides in length having about 18 to about 23(e.g., about 18, 19, 20, 21, 22, or 23) base pairs wherein the circularoligonucleotide forms a dumbbell shaped structure having about 19 basepairs and 2 loops.

A circular siNA molecule contains two loop motifs, wherein one or bothloop portions of the siNA molecule is biodegradable. For example, acircular siNA molecule of the invention is designed such thatdegradation of the loop portions of the siNA molecule in vivo cangenerate a double-stranded siNA molecule with 3′-terminal overhangs,such as 3′-terminal nucleotide overhangs comprising about 2 nucleotides.

Modified nucleotides present in siNA molecules, preferably in theantisense strand of the siNA molecules, but also optionally in the senseand/or both antisense and sense strands, comprise modified nucleotideshaving properties or characteristics similar to naturally occurringribonucleotides. For example, the invention features siNA moleculesincluding modified nucleotides having a Northern conformation (e.g.,Northern pseudorotation cycle, see for example Saenger, Principles ofNucleic Acid Structure, Springer-Verlag ed., 1984). As such, chemicallymodified nucleotides present in the siNA molecules of the invention,preferably in the antisense strand of the siNA molecules of theinvention, but also optionally in the sense and/or both antisense andsense strands, are resistant to nuclease degradation while at the sametime maintaining the capacity to mediate RNAi. Non-limiting examples ofnucleotides having a northern configuration include locked nucleic acid(LNA) nucleotides (e.g., 2′-O, 4′-C-methylene-(D-ribofuranosyl)nucleotides); 2′-methoxyethoxy (MOE) nucleotides; 2′-methyl-thio-ethyl,2′-deoxy-2′-fluoro nucleotides. 2′-deoxy-2′-chloro nucleotides, 2′-azidonucleotides, and 2′-O-methyl nucleotides.

The sense strand of a double stranded siNA molecule may have a terminalcap moiety such as an inverted deoxybasic moiety, at the 3′-end, 5′-end,or both 3′ and 5′-ends of the sense strand.

A siNA further may be further comprised of a nucleotide, non-nucleotide,or mixed nucleotide/non-nucleotide linker that joins the sense region ofthe siNA to the antisense region of the siNA. In one embodiment, anucleotide linker can be a linker of >2 nucleotides in length, forexample about 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides in length. Inanother embodiment, the nucleotide linker can be a nucleic acid aptamer.By “aptamer” or “nucleic acid aptamer” as used herein is meant a nucleicacid molecule that binds specifically to a target molecule wherein thenucleic acid molecule has sequence that comprises a sequence recognizedby the target molecule in its natural setting. Alternately, an aptamercan be a nucleic acid molecule that binds to a target molecule where thetarget molecule does not naturally bind to a nucleic acid. The targetmolecule can be any molecule of interest. For example, the aptamer canbe used to bind to a ligand-binding domain of a protein, therebypreventing interaction of the naturally occurring ligand with theprotein. This is a non-limiting example and those in the art willrecognize that other embodiments can be readily generated usingtechniques generally known in the art. [See, for example, Gold et al,Annu. Rev. Biochem., 64: 763 (1995); Brody and Gold, J. Biotechnol., 74:5 (2000); Sun, Curr. Opin. Mol. Ther., 2:100 (2000); Kusser, J.Biotechnol., 74: 27 (2000); Hermann and Patel, Science 287: 820 (2000);and Jayasena, Clinical Chemistry, 45: 1628. (1999)

A non-nucleotide linker may be comprised of an abasic nucleotide,polyether, polyamine, polyamide, peptide, carbohydrate, lipid,polyhydrocarbon, or other polymeric compounds (e.g. polyethylene glycolssuch as those having between 2 and 100 ethylene glycol units). Specificexamples include those described by Seela and Kaiser, Nucleic AcidsRes., 18:6353 (1990) and Nucleic Acids Res., 15:3113 (1987); Cload andSchepartz, J. Am. Chem. Soc., 113:6324 (1991); Richardson and Schepartz,J. Am. Chem. Soc., 113:5109 (1991); Ma et al., Nucleic Acids Res.,21:2585 (1993) and Biochemistry 32:1751(1993); Durand et al., NucleicAcids Res., 18:6353 (1990); McCurdy et al., Nucleosides & Nucleotides,10:287 (1991); Jschke et al., Tetrahedron Lett., 34:301 (1993); Ono etal., Biochemistry, 30:9914 (1991); Arnold et al., InternationalPublication No. WO 89/02439; Usman et al., International Publication No.WO 95/06731; Dudycz et al., International Publication No. WO 95/11910and Ferentz and Verdine, J. Am. Chem. Soc., 113:4000 (1991). A“non-nucleotide” further means any group or compound that can beincorporated into a nucleic acid chain in the place of one or morenucleotide units, including either sugar and/or phosphate substitutions,and allows the remaining bases to exhibit their enzymatic activity. Thegroup or compound can be abasic in that it does not contain a commonlyrecognized nucleotide base, such as adenosine, guanine, cytosine, uracilor thymidine, for example at the C1 position of the sugar.

The synthesis of a siNA molecule of the invention, which can bechemically-modified, comprises: (a) synthesis of two complementarystrands of the siNA molecule; (b) annealing the two complementarystrands together under conditions suitable to obtain a double-strandedsiNA molecule. In another embodiment, synthesis of the two complementarystrands of the siNA molecule is by solid phase oligonucleotidesynthesis. In yet another embodiment, synthesis of the two complementarystrands of the siNA molecule is by solid phase tandem oligonucleotidesynthesis.

Oligonucleotides (e.g., certain modified oligonucleotides or portions ofoligonucleotides lacking ribonucleotides) are synthesized usingprotocols known in the art, for example as described in Caruthers etal., 1992, Methods in Enzymology 211, 3-19, Thompson et al.,International PCT Publication No. WO 99/54459, Wincott et al., 1995,Nucleic Acids Res. 23, 2677-2684, Wincott et al., 1997, Methods Mol.Bio., 74, 59, Brennan et al., 1998, Biotechnol Bioeng., 61, 33-45, andBrennan, U.S. Pat. No. 6,001,311. Synthesis of RNA, including certainsiNA molecules of the invention, follows general procedures asdescribed, for example, in Usman et al., 1987, J. Am. Chem. Soc., 109,7845; Scaringe et al., 1990, Nucleic Acids Res., 18, 5433; and Wincottet al., 1995, Nucleic Acids Res. 23, 2677-2684 Wincott et al., 1997,Methods Mol. Bio., 74, 59.

Heterocyclic Drugs

Heterocyclic drugs, and particularly those containing at least onenitrogen heterocyclic ring can be employed as pharmaceutical agents inthe methods described herein. For example, yohimbine is an indolealkaloid that blocks alpha-2-adrenergic receptors. Its peripheraleffects are to increase cholinergic activity at the same time that itdecreases adrenergic activity. This combination has led to the use ofyohimbine in the treatment and diagnostic classification of certaintypes of male erectile impotence.

Other examples of heterocyclic drugs includes, but is not limited tomorphine, methotrexate (formerly Amethopterin,N-[4-[[(2,4-diamino-6-pteridinyl)-methyl]methylamino]benzoyl]-L-glutamicacid), Lorazepam(7-chloro-5-(o-chloro-phenyl)-1,3-dihydro-3-hydroxy-2H-1,4-benzodiazepin-2-one),6-Mercaptopurine, (1,7-dihydro-6H-purine-6-thione monohydrate),5-fluorouracil, nicotine, nicotinic acid and niacin.

Formulations and Delivery of Pharmaceutical Agents

The compositions of the present invention generally comprise a fusogenicsaposin protein or polypeptide, which is associated with an anionicliposome comprised of either at least one anionic long-chain lipid, withor without at least one neutral long chain lipid, and at least oneneutral or anionic short-chain lipids, containing a pharmaceutical orimaging agent in a safe and effective amount for the desired effect, allcontained in a pharmaceutically acceptable carrier with an appropriatepH. A safe and effective amount of the active agent is defined as anamount which would cause the desired cosmetic or therapeutic effect in apatient. An experienced practitioner, skilled in this invention wouldhave knowledge of the appropriate dosing ratios.

The appropriate dosage administered in any given case will, of course,vary depending upon known factors, such as the pharmacodynamiccharacteristics of the particular pharmaceutical agent, and its mode androute of administration; the age, general health, metabolism, weight ofthe recipient and other factors which influence response to thecompound; the kind of concurrent treatment, the frequency of treatment,and the effect desired.

In one embodiment, the invention comprises a desired pharmaceuticalagent, in a safe and effective amount, which is incorporated intoanionic liposomes, in a buffered aqueous solution of a pH of about 5.5or less. The preferred fusogenic protein or polypeptide is saposin C, inconcentrations from about 20 nM to about 100 nM (nanomolar), preferablyabout 40 to about 50 nM, which is then introduced to theliposome-pharmaceutical agent mixture. The concentration of theliposomes is in excess to that of the fusogenic protein or polypeptideand is about a 1 to 10-fold excess, by molar ratio, or about a 3 to 7fold excess to that of saposin C (i.e. at least a 1:10 by molar ratio ofsaposin C:liposome). In this embodiment, at least one imaging agenthaving at least one imaging property may be added to the liposomalcomposition. Alternatively, in this embodiment, the pharmaceutical agentmay be substituted with the imaging agent.

In one embodiment, the liposome contains at least one type of negativelycharged long-chain lipid such as dioleoylphosphatidyserine (DOPS). Theliposomes may be made from any mixture of lipids that contain a suitableamount of anionic long-chain lipids. In one particular embodiment, theliposomes are made from a mixture containing anionic long-chain lipids(such as DOPS or dimyristoyl phosphatidylglcerol (DMPG)), neutrallong-chain lipids (such as dipalmitoyl phosphatidylcholine (DPPC) ordimyristoyl phosphatidylcholine (DMPC)), and neutral short-chain lipids(such as DHPC). The overall charge of the resulting liposome derivedfrom the mixture of lipids is negative. The short chain phospholipidsmay also be negatively charged.

Such a composition could then be applied topically to the skin oradministered to other tissues or the brain and CNS via the methodsdescribed herein. Other examples of preparing such liposome-fusionprotein complexes, in which an active agent is contained within theliposome, are given in U.S. Pat. No. 6,099,857, Gross, Aug. 8, 2000 andU.S. Pat. No. 5,766,626, Gross, Jun. 16, 1998, which are hereinincorporated by reference.

Transdermal Delivery

The pharmaceutical agent-chemical modifier complexes described hereincan be administered transdermally. Transdermal administration typicallyinvolves the delivery of a pharmaceutical agent for percutaneous passageof the drug into the systemic circulation of the patient. The skin sitesinclude anatomic regions for transdermally administering the drug andinclude the forearm, abdomen, chest, back, buttock, mastoidal area, andthe like.

Transdermal delivery is accomplished by exposing a source of the complexto a patient's skin for an extended period of time. Transdermal patcheshave the added advantage of providing controlled delivery of apharmaceutical agent to the body (see Transdermal Drug Delivery:Developmental Issues and Research Initiatives, Hadgraft and Guy (eds.),Marcel Dekker, Inc., (1989); Controlled Drug Delivery: Fundamentals andApplications, Robinson and Lee (eds.), Marcel Dekker Inc., (1987); andTransdermal Delivery of Drugs, Vols. 1-3, Kydonieus and Berner (eds.),CRC Press, (1987)). Such dosage forms can be made by dissolving,dispersing, or otherwise incorporating the pharmaceutical agent, saposinC and anionic liposomes in a proper medium, such as an elastomericmatrix material. Absorption enhancers can also be used to increase theflux of the compound across the skin. The rate of such flux can becontrolled by either providing a rate-controlling membrane or dispersingthe compound in a polymer matrix or gel.

Passive Transdermal Drug Delivery

A variety of types of transdermal patches will find use in the methodsdescribed herein. For example, a simple adhesive patch can be preparedfrom a backing material and an acrylate adhesive. The pharmaceuticalagent-chemical modifier complex and any enhancer are formulated into theadhesive casting solution and allowed to mix thoroughly. The solution iscast directly onto the backing material and the casting solvent isevaporated in an oven, leaving an adhesive film. The release liner canbe attached to complete the system.

Alternatively, a polyurethane matrix patch can be employed to deliverthe pharmaceutical agent-chemical modifier complex. The layers of thispatch comprise a backing, a polyurethane drug/enhancer matrix, amembrane, an adhesive, and a release liner. The polyurethane matrix isprepared using a room temperature curing polyurethane prepolymer.Addition of water, alcohol, and complex to the prepolymer results in theformation of a tacky firm elastomer that can be directly cast only thebacking material.

A further embodiment of this invention will utilize a hydrogel matrixpatch. Typically, the hydrogel matrix will comprise alcohol, water,drug, and several hydrophilic polymers. This hydrogel matrix can beincorporated into a transdermal patch between the backing and theadhesive layer.

For passive delivery systems, the rate of release is typicallycontrolled by a membrane placed between the reservoir and the skin, bydiffusion from a monolithic device, or by the skin itself serving as arate-controlling barrier in the delivery system (see U.S. Pat. Nos.4,816,258; 4,927,408; 4,904,475; 4,588,580, 4,788,062). The rate of drugdelivery will be dependent, in part, upon the nature of the membrane.For example, the rate of drug delivery across membranes within the bodyis generally higher than across dermal barriers. The rate at which thecomplex is delivered from the device to the membrane is mostadvantageously controlled by the use of rate-limiting membranes whichare placed between the reservoir and the skin. Assuming that the skin issufficiently permeable to the complex (i.e., absorption through the skinis greater than the rate of passage through the membrane), the membranewill serve to control the dosage rate experienced by the patient.

Suitable permeable membrane materials may be selected based on thedesired degree of permeability, the nature of the complex, and themechanical considerations related to constructing the device. Exemplarypermeable membrane materials include a wide variety of natural andsynthetic polymers, such as polydimethylsiloxanes (silicone rubbers),ethylenevinylacetate copolymer (EVA), polyurethanes,polyurethane-polyether copolymers, polyethylenes, polyamides,polyvinylchlorides (PVC), polypropylenes, polycarbonates,polytetrafluoroethylenes (PTFE), cellulosic materials, e.g., cellulosetriacetate and cellulose nitrate/acetate, and hydrogels, e.g.,2-hydroxyethylmethacrylate (HEMA).

Other items may be contained in the device, such as other conventionalcomponents of therapeutic products, depending upon the desired devicecharacteristics. For example, the compositions according to thisinvention may also include one or more preservatives or bacteriostaticagents, e.g., methyl hydroxybenzoate, propyl hydroxybenzoate,chlorocresol, benzalkonium chlorides, and the like. These pharmaceuticalcompositions also can contain other active ingredients such asantimicrobial agents, particularly antibiotics, anesthetics, analgesics,and antipruritic agents.

Topical Treatments

Another aspect of this invention provides for the topical delivery ofpharmaceutical compositions. This treatment regimen is suitable eitherfor the systemic administration of the pharmaceutical agent or forlocalized therapy, i.e., directly to pathological or diseased tissue.

Typically, the topical formulations will comprise a preparation fordelivering the pharmaceutical agent-chemical modifier complex directlyto the affected skin comprising the complex, typically in concentrationsin the range of from about 0.001% to 10%; preferably, from about 0.01 toabout 10%; more preferably, from about 0.1 to about 5%; and mostpreferably, from about 1 to about 5%, together with a non-toxic,pharmaceutically acceptable topical carrier (see DermatologicalFormulations: Percutaneous Absorption, Barry (ed.), Marcel Dekker Inc.,(1983); for standard dosages of conventional pharmaceutical agents, see,e.g., Physicians Desk Reference (1992 Edition); and American MedicalAssociation (1992) Drug Evaluations Subscriptions).

Topical preparations can be prepared by combining the pharmaceuticalagent-chemical modifier complex with conventional pharmaceuticaldiluents and carriers commonly used in topical dry, liquid, cream andaerosol formulations. Ointment and creams may, for example, beformulated with an aqueous or oily base with the addition of suitablethickening and/or gelling agents. Such bases may include water and/or anoil such as liquid paraffin or a vegetable oil such as peanut oil orcastor oil. Thickening agents which may be used according to the natureof the base include soft paraffin, aluminum stearate, cetostearylalcohol, propylene glycol, polyethylene glycols, woolfat, hydrogenatedlanolin, beeswax, and the like. Lotions may be formulated with anaqueous or oily base and will, in general, also include one or more ofthe following: stabilizing agents, emulsifying agents, dispersingagents, suspending agents, thickening agents, coloring agents, perfumes,and the like. Powders may be formed with the aid of any suitable powderbase, e.g., talc, lactose, starch, and the like. Drops may be formulatedwith an aqueous base or non-aqueous base also comprising one or moredispersing agents, suspending agents, solubilizing agents, and the like.

Dosage forms for the topical administration of a complex of thisinvention include powders, sprays, ointments, pastes, creams, lotions,gels, solutions, patches and inhalants. The active compound may be mixedunder sterile conditions with a pharmaceutically-acceptable carrier, andwith any preservatives, buffers, or propellants which may be required.

The ointments, pastes, creams and gels also may contain excipients, suchas animal and vegetable fats, oils, waxes, paraffins, starch,tragacanth, cellulose derivatives, polyethylene glycols, silicones,bentonites, talc and zinc oxide, or mixtures thereof. Powders and spraysalso can contain excipients such as lactose, talc, aluminum hydroxide,calcium silicates and polyamide powder, or mixtures of these substances.Sprays can additionally contain customary propellants, such aschlorofluorohydrocarbons and volatile unsubstituted hydrocarbons, suchas butane and propane.

Transmucosal Delivery

Although much of the discussion herein has centered on techniques fortransdermal delivery, the methods of the present invention are alsoapplicable to the enhanced transport and delivery of pharmaceuticalagents through mucosal membranes, such as gastrointestinal, sublingual,buccal, nasal, pulmonary, vaginal, corneal, and ocular membranes (seeMackay et al. (1991) Adv. Drug Del. Rev, 7:313-338). Specifically, thereare many similarities between skin and mucosal membranes. For example,the membrane of the buccal cavity is non-keratinized. However, thebuccal membrane is similar to the skin because both are stratified withthe former consisting of polygonal cells at the basal membrane leadingto squamous cells at the surface.

Transmucosal (i.e., sublingual, buccal and vaginal) drug deliveryprovides for an efficient entry of active substances to systemiccirculation and reduce immediate metabolism by the liver and intestinalwall flora. Transmucosal drug dosage forms (e.g., tablet, suppository,ointment, gel, pessary, membrane, and powder) are typically held incontact with the mucosal membrane and disintegrate and/or dissolverapidly to allow immediate systemic absorption.

Buccal Administration

For delivery to the buccal or sublingual membranes, typically an oralformulation, such as a lozenge, tablet, or capsule will be used. Themethod of manufacture of these formulations are known in the art,including but not limited to, the addition of the pharmaceuticalagent-chemical modifier complex to a pre-manufactured tablet; coldcompression of an inert filler, a binder, and either a pharmaceuticalagent-chemical modifier complex or a substance containing the complex(as described in U.S. Pat. No. 4,806,356 incorporated by reference) andencapsulation.

Another oral formulation is one that can be applied with an adhesive,such as the cellulose derivative, hydroxypropyl cellulose, to the oralmucosa, for example as described in U.S. Pat. No. 4,940,587,incorporated by reference. This buccal adhesive formulation, whenapplied to the buccal mucosa, allows for controlled release of thepharmaceutical agent-chemical modifier complex into the mouth andthrough the buccal mucosa.

Nasal/Pulmonary Administration

For delivery to the nasal and/or pulmonary membranes, typically anaerosol formulation will be employed. The term “aerosol” includes anygas-borne suspended phase of the pharmaceutical agent-chemical modifiercomplex which is capable of being inhaled into the bronchioles or nasalpassages. Specifically, aerosol includes a gas-borne suspension ofdroplets of the compounds of the instant invention, as may be producedin a metered dose inhaler or nebulizer, or in a mist sprayer. Aerosolalso includes a dry powder composition of the pharmaceuticalagent-chemical modifier complex suspended in air or other carrier gas,which may be delivered by inhalation from an inhaler device.

Delivery Across the Blood-Brain Barrier

The present invention can also be used to transport pharmaceutical orimaging agents across the blood-brain barrier. Liposomes containingsaposin C (or a variant or peptide thereof) and a negatively chargedlong-chain lipid such as DOPS can be administered intramuscularly,intravenously, intraocularly or transnasally for delivery to the CNS,specifically the brain, using methods as described in the art.Administration via the nasal cavity, for example, results in entry intothe olfactory CSF then into the peripheral bloodstream similar to anintracerebroventricular infusion (ICV). As describe above in full, itshould be understood by one skilled in the art that other, neutrallong-chain lipids and/or short-chain lipids (neutral or negative) may beincluded in the composition of the liposome as described above toimprove stability or utility of the final composition

As an example of successful transport into the CNS, the inventor hasdemonstrated that saposin C can be transported into the cultured mousecortical and hippocampal neurons, facilitated by complexes with DOPSliposomes. Saposin C can be transported into endosomal and lysosomalcompartments using saposin-C liposomes containing long-chain anionicphospholipids. This method can be used for the treatment of neurologicaldiseases including, for example, those in which MVB accumulationcontributes to pathology and progression of disease. For example, inPSAP−/− mice, in which MVB formation is found in neurons and braintissues, administration of the DOPS-saposin C liposomes via tailinjection resulted in a reduction of accumulation of these structures.See FIGS. 5 and 7.

In another embodiment, a number of blood-brain targeting agents areconjugated to the surface of the liposome. Suitable targeting agentsinclude insulin, transferrin, insulin-like growth factor, or leptin, asthese peptides all have endogenous RMT systems within the BBB that alsoexist on the BCM, and these endogenous peptides could be used as“transportable peptides.” Alternatively, the surface of the liposomecould be conjugated with 2 different “transportable peptides,” onepeptide targeting an endogenous BBB receptor and the other targeting anendogenous BCM peptide. The latter could be specific for particularcells within the brain, such as neurons, glial cells, pericytes, smoothmuscle cells, or microglia. Targeting peptides may be endogenous peptideligands of the receptors, analogues of the endogenous ligand, orpeptidomimetic MAbs that bind the same receptor of the endogenousligand. The use of transferrin receptor (TfR)-specific peptidomimeticmonoclonal antibodies as BBB “transportable peptides” are described indetail in U.S. Pat. Nos. 5,154,924; 5,182,107; 5,527,527; 5,672,683;5,833,988; and 5,977,307. The use of an MAb to the human insulinreceptor (HIR) as a BBB “transportable peptide” has been described.

The conjugation agents which are used to conjugate the blood-barriertargeting agents to the surface of the liposome can be any of thewell-known polymeric conjugation agents such as sphingomyelin,polyethylene glycol (PEG) or other organic polymers. In one embodiment,PEG is the conjugation agent. In one embodiment, the molecular weight ofthe conjugation agent is between 1000 and 50,000 DA. In one embodiment,the conjugation agent is a bifunctional 2000 DA PEG which contains alipid at one end and a maleimide group at the other end. The lipid endof the PEG binds to the surface of the liposome with the maleimide groupbonding to the receptor-specific monoclonal antibody or otherblood-brain barrier targeting vehicle. In one embodiment, from 5 to 1000targeting vehicles is conjugated to each liposome. Liposomes havingapproximately 25-40 targeting vehicles conjugated thereto are providedin one embodiment.

Although the invention has been described using liposomes as thepreferred nanocontainer, it will be recognized by those skilled in theart that other nanocontainers may be used. For example, the liposome canbe replaced with a nanoparticle or any other molecular nanocontainerwith a diameter<200 nm that can encapsulate the DNA and protect thenucleic acid from nucleases while the formulation is still in the bloodor in transit from the blood to the intracellular compartment of thetarget cell. Also, the PEG strands can be replaced with multiple otherpolymeric substances such as sphingomylein, which are attached to thesurface of the liposome or nanocontainer and serve the dual purpose ofproviding a scaffold for conjugation of the “transportable peptide” andfor delaying the removal of the formulation from blood and optimizingthe plasma pharmacokinetics. Further, the present invention contemplatesdelivery of genes to any group of cells or organs which have specifictarget receptors.

Treatment of Gaucher Disease with Fusogenic Saposin Proteins andPolypeptides

Additionally, saposin C is essential for hydrolysis of glucosylceramidesto ceramide in vivo. A deficiency in epidermal glucocerebrosidaseresults in an altered glucosylceramide to ceramide ratio and thisaltered ratio is associated with skin barrier abnormalitiescharacterized by Gaucher Disease. It is thought that saposin C iscritical to the formation of the epidermal permeability barrier bymaintaining physiologic concentrations of glucosylceramide and ceramidein the stratum corneum. According to this model, the role of saposin Cin stimulating glucocerebrosidase is mediated by its destabilizingeffect on the membranes. Thus, in patients with epidermalglucocerebrosidase deficiency, a topical application of a saposinC-liposome complex, wherein the liposome contains acid beta glucosidase,the mixture contained in a pharmaceutically acceptable carrier may beused to fuse cell membranes in order to facilitate the hydrolysis ofglucosylceramide to ceramide to aid in regulation of skin barrierformation and function. These compositions can, for example, beformulated as creams, lotions, solutions or gels. The carrier mayinclude, for example, pharmaceutically acceptable emollients,emulsifiers, thickening agents, solvents, preservatives, coloring agentsand fragrances.

Saposin C Lysosomes as a Delivery System for Administration of ImagingAgents

In another embodiment of the present invention, the saposin C-containingliposome may be used to simultaneously deliver at least one imagingagent having one or more distinct imaging properties. These agents mayuse magnetic resonance imaging, fluorescence, or CT/PET detectionproperties. One or more imaging agents may be simultaneously integratedor encapsulated into the saposin-C-containing liposomes, such that asingle population of saposin-C-containing liposomes may be used todeliver multiple imaging agents, with or without a pharmaceutical agent,to the desired tissues.

In a further embodiment of the present invention, the liposome basedcontrast medium of the invention may further comprise additionalcontrast agents such as conventional contrast agents, which may serve toincrease the efficacy of the contrast medium for MRI. Many such contrastagents are well known to those skilled in the art and includeparamagnetic and superparamagnetic contrast agents.

Exemplary paramagnetic contrast agents suitable for use in the subjectinvention include stable free radicals (such as, for example, stablenitroxides), as well as compounds comprising transition, lanthanide andactinide elements, which may, if desired, be in the form of a salt ormay be covalently or noncovalently bound to complexing agents (includinglipophilic derivatives thereof) or to proteinaceous macromolecules.

Preferable transition, lanthanide and actinide elements include Gd(III),Mn(II), Cu(II), Cr(III), Fe(II), Fe(III), Co(II), Er(II), Ni(II),Eu(III) and Dy(III). More preferably, the elements include Gd(III),Mn(II), Cu(II), Fe(II), Fe(III), Eu(III) and Dy(III), especially Mn(II)and Gd(III).

These elements may, if desired, be in the form of a salt, such as amanganese salt, e.g., manganese chloride, manganese carbonate, manganeseacetate, and organic salts of manganese such as manganese gluconate andmanganese hydroxylapatite; and such as an iron salt, e.g., iron sulfidesand ferric salts such as ferric chloride.

These elements may also, if desired, be bound, e.g., covalently ornoncovalently, to complexing agents (including lipophilic derivativesthereof) or to proteinaceous macromolecules. Preferable complexingagents include, for example, diethylenetriamine-pentaacetic acid (DTPA),ethylene-diaminetetraacetic acid (EDTA),1,4,7,10-tetraazacyclododecane-N,N′,N′,N″-tetraacetic acid (DOTA),1,4,7,10-tetraazacyclododecane-N,N′,N″-triacetic acid (DO3A),3,6,9-triaza-12-oxa-3,6,9-tricarboxymethylene-10-carboxy-13-phenyl-tridecanoic acid (B-19036), hydroxybenzylethylene-diamine diacetic acid (HBED),N,N′-bis(pyridoxyl-5-phosphate)ethylene diamine, N,N′-diacetate (DPDP),1,4,7-triazacyclononane-N,N′,N″-triacetic acid (NOTA),1,4,8,11-tetraazacyclotetradecane-N,N′N″,N′″-tetraacetic acid (TETA),kryptands (that is, macrocyclic complexes), and desferrioxamine. Morepreferably, the complexing agents are EDTA, DTPA, DOTA, DO3A andkryptands, most preferably DTPA. Preferable lipophilic complexes thereofinclude alkylated derivatives of the complexing agents EDTA, DOTA, etc.,for example, EDTA-DDP, that is,N,N′-bis-(carboxy-decylamidomethyl-N-2,3-dihydroxypropyl)-ethylenediamine-N,N′-diacetate;EDTA-ODP, that isN,N′-bis-(carboxy-octadecylamido-methyl-N-2,3-dihydroxypropyl)-ethylenediamine-N,N′-diacetate; EDTA-LDPN,N′-Bis-(carboxy-laurylamidomethyl-N-2,3-dihydroxypropyl)-ethylenediamine-N,N′-diacetate;etc.; such as those described in U.S. Ser. No. 887,290, filed May 22,1992, the disclosures of which are hereby incorporated herein byreference in its entirety. Preferable proteinaceous macromoleculesinclude albumin, collagen, polyarginine, polylysine, polyhistidine,.gamma-globulin and beta-globulin. More preferably, the proteinaceousmacromolecules comprise albumin, polyarginine, polylysine, andpolyhistidine.

Suitable complexes thus include Mn(II)-DTPA, Mn(II)-EDTA, Mn(II)-DOTA,Mn(II)-DO3A, Mn(II)-kryptands, Gd(III)-DTPA, Gd(III)-DOTA, Gd(III)-DO3A,Gd(III)-kryptands, Cr(III)-EDTA, Cu(II)-EDTA, or iron-desferrioxamine,especially Mn(II)-DTPA or Gd(III)-DTPA.

Nitroxides are paramagnetic contrast agents which increase both T1 andT2 relaxation rates by virtue of one unpaired electron in the nitroxidemolecule. The paramagnetic effectiveness of a given compound as an MRIcontrast agent is at least partly related to the number of unpairedelectrons in the paragmagnetic nucleus or molecule, specifically to thesquare of the number of unpaired electrons. For example, gadolinium hasseven unpaired electrons and a nitroxide molecule has only one unpairedelectron; thus gadolinium is generally a much stronger MRI contrastagent than a nitroxide. However, effective correlation time, anotherimportant parameter for assessing the effectiveness of contrast agents,confers potential increased relaxivity to the nitroxides. When theeffective correlation time is very close to the proton Larmourfrequency, the relaxation rate may increase dramatically. When thetumbling rate is slowed, e.g., by attaching the paramagnetic contrastagent to a large structure, it will tumble more slowly and thereby moreeffectively transfer energy to hasten relaxation of the water protons.In gadolinium, however, the electron spin relaxation time is rapid andwill limit the extent to which slow rotational correlation times canincrease relaxivity. For nitroxides, however, the electron spincorrelation times are more favorable and tremendous increases inrelaxivity may be attained by slowing the rotational correlation time ofthese molecules. The liposomes of the present invention are ideal forattaining the goals of slowed rotational correlation times and resultantimprovement in relaxivity. Although not intending to be bound by anyparticular theory of operation, it is contemplated that since thenitroxides may be designed to coat the perimeters of the liposomes,e.g., by making alkyl derivatives thereof, that the resultingcorrelation times can be optimized. Moreover, the resulting contrastmedium of the present invention may be viewed as a magnetic sphere, ageometric configuration which maximizes relaxivity.

If desired, the nitroxides may be alkylated or otherwise derivitized,such as the nitroxides 2,2,5,5-tetramethyl-1-pyrrolidinyloxy, freeradical, and 2,2,6,6-tetramethyl-1-piperidinyloxy, free radical (TMPO),

Exemplary superparamagnetic contrast agents suitable for use in thesubject invention include metal oxides and sulfides which experience amagnetic domain, ferro- or ferrimagnetic compounds, such as pure iron,magnetic iron oxide (such as magnetite), .gamma-Fe2 O3, manganeseferrite, cobalt ferrite and nickel ferrite.

The contrast agents, such as the paramagnetic and superparamagneticcontrast agents described above, may be employed as a component withinthe microspheres or in the contrast medium comprising the microspheres.They may be entrapped within the internal space of the microspheres,administered as a solution with the microspheres or incorporated intothe stabilizing compound forming the microsphere wall.

For example, if desired, the paramagnetic or superparamagnetic agentsmay be delivered as alkylated or other derivatives incorporated into thestabilizing compound, especially the lipidic walls of the microspheres.In particular, the nitroxides 2,2,5,5-tetramethyl-1-pyrrolidinyloxy,free radical and 2,2,6,6-tetramethyl-1-piperidinyloxy, free radical, canform adducts with long chain fatty acids at the positions of the ringwhich are not occupied by the methyl groups, via a number of differentlinkages, e.g., an acetyloxy group. Such adducts are very amenable toincorporation into the stabilizing compounds, especially those of alipidic nature, which form the walls of the microspheres of the presentinvention.

Mixtures of any one or more of the paramagnetic agents and/orsuperparamagnetic agents in the contrast media may similarly be used.

The paramagnetic and superparamagnetic agents described above may alsobe coadministered separately, if desired.

The liposomes used in the present invention may not only serve aseffective carriers of the superparamagnetic agents, e.g., iron oxides,but also appear to magnify the effect of the susceptibility contrastagents. Superparamagnetic contrast agents include metal oxides,particularly iron oxides but including manganese oxides, and as ironoxides, containing varying amounts of manganese, cobalt and nickel whichexperience a magnetic domain. These agents are nano or microparticlesand have very high bulk susceptibilities and transverse relaxationrates. The larger particles, e.g., 100 nm diameter, have much higher R2relaxivities than R1 relaxivities but the smaller particles, e.g., 10 to15 nm diameter have somewhat lower R2 relaxivities, but much morebalanced R1 and R2 values. The smallest particles, e.g., monocrystallineiron oxide particles, 3 to 5 nm in diameter, have lower R2 relaxivities,but probably the most balanced R1 and R2 relaxation rates. Ferritin canalso be formulated to encapsulate a core of very high relaxation ratesuperparamagnetic iron. It has been discovered that stabilized liposomesused in the present invention can increase the efficacy and safety ofthese conventional iron oxide based MRI contrast agents.

Incorporation of imaging agents into liposomes is advantageous fordetermining uptake and delivery of the pharmaceutical agent containedtherein. Further, such agents can also permit the imaging of tissuestructure, or in the case of cancers, the extent of metastasis or tumorgrowth. In one embodiment of the present invention, saposin-C-containingliposomes can transfer both pharmaceutical and imaging agents acrossbiological membranes. In another embodiment, multiple imaging agents canbe incorporated into the liposomal membrane, or, imaging agents havingmultiple imaging properties (such as the PTIR agents described above andin the Examples) can be used. Either method allows the clinician orresearcher to utilize multiple methods of detection with a singleadministration of the liposomal composition.

Imaging agents may use magnetic resonance imaging, fluorescence orPT/CAT devices. The use of magnetic resonance imaging (MRI) contrastenhancement agents or radioactive isotopes in the body is practiced by avariety of methods. For example, Li, et al., U.S. Pat. No. 6,569,451,incorporated herein by reference, teaches a method by which polymerizedliposome particles may be used to deliver contrast agents such as thoseusing magnetic resonance imaging.

In one embodiment of the present invention, MR contrast agents (such asUltrasmall SuperParamagnetic Iron Oxide (USPIO) nanoparticles) can beencapsulated within the aqueous interior of the liposome. MRI scanningmay employ chelates of gadolinium or manganese. However, labeling ofnon-phagocytic cells for MR detection requires that the liposomesencapsulate and deliver sufficient quantities of the contrast agent.Tumor-specific liposomes can be used to deliver the agent to the tissue,aiding in earlier detection and better visualization using MRI. Deliveryand uptake of targeted drugs can also be estimated using contrastenhanced MR microimaging, by using liposomes of the present invention asdual carriers for the drug and the contrast agent. For example, COMBIDEX(Advanced Magnetics, MA, size of 0 nm) a molecular imaging agentdetected using MRI can be encapsulated in liposomes made ofdioleylphosphatidyserine (DOPS). These liposomes can then be effectivelydelivered to human neuroblastoma cells. This is described in detail inExample 3 of the present invention.

If desired, two or more different ions may be used in combination. Asthose skilled in the art will recognize, once armed with the presentdisclosure, various combinations of the lipsoluble compounds andparamagnetic ions may be used to modify the relaxation behavior of theresulting contrast agent. The subject paramagnetic ion and liposolublecompound complexes of the invention have been found to be extremelyeffective contrast enhancement agents for magnetic resonance imaging.

The liposoluble compounds of the present invention may be employedsinglely or in combination with one another, and in combination with oneor more paramagnetic ions as contrast agents for magnetic resonanceimaging. Exemplary paramagnetic ions include transition, lanthanide(rare earth) and actinide ions, as will be readily apparent to thoseskilled in the art, in view of the present disclosure. Preferableparamagnetic ions include those selected from the group consisting ofCr3, Co2, Mn2, Ni2, Fe3, Fe2, La3, Cu2, Gd3, Ce3, Tb3, Pr3, Dy3, Nd3,Ho3, Pm3, Er3, Sm3, Tm3, Eu3, Yb3 and Lu3. More preferably, theparamagnetic ion is selected from the group consisting of Mn2, Fe3 andGd3, most preferably Mn2.

Multiple contrast agents are available for enhancing tissue contrast inmagnetic resonance Imaging. Some of the most commonly used contrastagents are chelates of Gadolinium, such as Gd-DTPA, Gd-DTPA-BMA, andGd-DOTA. Most currently available contrast agent formulations are ofsmall molecular size. In one embodiment, the contrast agent is selectedfrom the group consisting of iodine, gadolinium and magnetite.

Additionally, fluorescent imaging agents may be incorporated within theliposomes of the present invention, thus providing an additional meansof detection. For example, NBD, Rhodamine, the PTIR labels describedabove, or other known fluorescent agents may be used. Any commerciallyavailable fluorescent label or fluorescently labeled dye (eitherlipophilic or containing a lipophilic moiety) such as those describedabove may be used with the present invention. Hui, L. et al. describesmethods wherein the PTIR contrast agents can be used to label LDLparticles, and is incorporated herein by reference. Hui, L. et al., MRand Fluroescent Imaging of Low Density Lipoproteing Receptors, AcadRadiol 2004; 11:1251-1259. The total concentration of the fluorescentagent in the lipid composition is about 1% to about 5% or about 2% toabout 4%. Of the fluorescent agents, markers emitting longer wavelengths(red fluorescence) such as PTIR 271 and 316 yield less background invivo. Blue and green wavelengths have greater background signal. PTIR271 has been demonstrated by the inventors to incorporate intosaposin-C-containing liposomes with minimal background and clearlydetectable signal. FIG. 7 illustrates uptake of DOPS liposomescontaining PTIR 271 and 316.

Liquid, iodine-containing compounds, suitably iodo- or polyiodophenylderivatives, are used as iodine-containing contrast agents. Suitablematerials include Iopromide, Ioxitalamate, Ioxaglate, Iopamidol,Iohexol, Iotralon, Metrizamide or Ultravist. At the same time, thecontrasting agent serves as solvent for the mixture of thelyophilisates. Either gadolinium- or magnetite-containing contrastingagents are used for the magnetic resonance tomography (MRT). Suitablyfrom 30 mg to 90 mg lyophilized particles are mixed in the required.amounts of cytostatic drug and subsequently dissolved in from 3 ml to 6ml of contrasting agent.

The new preparation and its use enable without the help of indirectmethods, using X-ray fluoroscopy, a sufficient embolization beingportrayed directly, the tumor with its blood vessels being imaged as astill picture; using gadolinium- or magnetite-containing contrast drugsin combination with flow-coded measurement sequences, and theembolization can also be portrayed with the help of magnetic resonance.tomography; the attainable concentration of cytostatic drugs in thetumor tissue is considerably increased (by up to a factor of 20) incomparison to other forms of administration; and the application issimplified while, at the same time, the safety is increased (retrogradefaulty perfusion is avoided).

Finally, imaging agents that use computed tomography (CT scan) orpositron emission tomography (PET) can be used. The most commonlyemployed radionuclide imaging agents include radioactive iodine andindium. Imaging by CT scan may employ a heavy metal such as ironchelates. Additionally, positron emission tomography (PET) may bepossible using positron emitters of oxygen, nitrogen, iron, carbon, orgallium. Example of radionuclides useful in imaging procedures include:⁴³K, ⁵²Fe, ⁵⁷Co, ⁶⁷Cu, ⁶⁷Ga, ⁶⁸Ga, ⁷⁷Br, ⁸¹Rb, ⁸¹Kr, ⁸⁷Sr, ⁹⁹Tc, ¹¹¹In,¹¹³In, ¹²³I, ¹²⁵I, ¹²⁷Cs, ¹²⁹Cs, ¹³¹I, ¹³²I, ¹⁹⁷Hg, ²⁰³Pb and ²⁰⁶Bi.These imaging agents detectable by CT/PET can be incorporated into thesaposin-C liposome using methods known by those skilled in the art.

One having ordinary skill in the art may conjugate a saposin-Cpolypeptide to a radionuclide using well-known techniques. For example,Magerstadt, M. (1991) Antibody Conjugates And Malignant Disease, CRCPress, Boca Raton, Fla.; and Barchel, S. W. and Rhodes, B. H., (1983)Radioimaging and Radiotherapy, Elsevier, New York, N.Y., each of whichis incorporated herein by reference, teach the conjugation of varioustherapeutic and diagnostic radionuclides to amino acids of antibodies.Such reactions may be applied to conjugate radionuclides to saposin-Cpeptides or to saposin-C peptides with an appropriate linker.

Labels

The compositions of this invention optionally include one or morelabels; e.g., optically detectable labels, such as fluorescent orluminescent labels, and/or non-optically detectable labels, such asmagnetic labels. A number of fluorescent labels are well known in theart, including but not limited to, quantum dots, hydrophobicfluorophores (e.g., coumarin, rhodamine and fluorescein), and greenfluorescent protein (GFP) and variants thereof (e.g., cyan fluorescentprotein and yellow fluorescent protein). See e.g., Haughland (2002)Handbook of Fluorescent Probes and Research Products, Ninth Edition orthe current Web Edition, both available from Molecular Probes, Inc.Likewise, a variety of donor/acceptor and fluorophore/quenchercombinations, using e.g., fluorescence resonance energy transfer(FRET)-based quenching, non-FRET based quenching, or wavelength-shiftingharvester molecules, are known. Example combinations include cyanfluorescent protein and yellow fluorescent protein, terbium chelate andTRITC (tetrarhodamine isothiocyanate), lanthanide (e.g., europium orterbium) chelates and allophycocyanin (APC) or Cy5, europium cryptateand Allophycocyanin, fluorescein and tetramethylrhodamine, IAEDANS andfluorescein, EDANS and DABCYL, fluorescein and DABCYL, fluorescein andfluorescein, BODIPY FL and BODIPY FL, and fluorescein and QSY 7 dye.Nonfluorescent acceptors such as DABCYL and QSY 7 and QSY 33 dyes havethe particular advantage of eliminating background fluorescenceresulting from direct (i.e., nonsensitized) acceptor excitation. See,e.g., U.S. Pat. Nos. 5,668,648, 5,707,804, 5,728,528, 5,853,992, and5,869,255 to Mathies et al. for a description of FRET dyes.

For use of quantum dots as labels for biomolecules, see, e.g., Dubertretet al. (2002) Science 298:1759; Nature Biotechnology (2003) 21:41-46;and Nature Biotechnology (2003) 21:47-51. In the context of the presentinvention, such quantum dots can be used to label any nucleic acid ofinterest, e.g., an interfering RNA.

Other optically detectable labels can also be used in the invention. Forexample, gold beads can be used as labels and can be detected using awhite light source via resonance light scattering. See, e.g.,http://www.geniconsciences.com. Suitable non-optically detectable labelsare also known in the art. For example, magnetic labels can be used inthe invention (e.g., 3 nm superparamagnetic colloidal iron oxide as alabel and NMR detection; see e.g., Nature Biotechnology (2002)20:816-820).

Labels can be introduced to nucleic acids during synthesis or bypostsynthetic reactions by techniques established in the art. Forexample, a fluorescently labeled nucleotide can be incorporated into anRNA or DNA during enzymatic or chemical synthesis of the nucleic acid,e.g., at a preselected or random nucleotide position. Alternatively,fluorescent labels can be added to RNAs or DNAs by postsyntheticreactions, at either random or preselected positions (e.g., anoligonucleotide can be chemically synthesized with a terminal amine orfree thiol at a preselected position, and a fluorophore can be coupledto the oligonucleotide via reaction with the amine or thiol). Reagentsfor fluorescent labeling of nucleic acids are commercially available;for example, a variety of kits for fluorescently labeling nucleic acidsare available from Molecular Probes, Inc. (www.probes.com), and a kitfor randomly labeling double-stranded RNA is available from Ambion, Inc.(www.ambion.com, the Silencer™ siRNA labeling kit). Quenchers can beintroduced by analogous techniques.

Attachment of labels to oligos during automated synthesis and bypost-synthetic reactions has been described. See, e.g., Tyagi and Kramer(1996) “Molecular beacons: probes that fluoresce upon hybridization”Nature Biotechnology 14:303-308; U.S. Pat. No. 6,037,130 to Tyagi et al.(Mar. 14, 2000), entitled “Wavelength-shifting probes and primers andtheir use in assays and kits”; and U.S. Pat. No. 5,925,517 (Jul. 20,1999) to Tyagi et al. entitled “Detectably labeled dual conformationoligonucleotide probes, assays and kits.” Additional details onsynthesis of functionalized oligos can be found in Nelson, et al. (1989)“Bifunctional Oligonucleotide Probes Synthesized Using A Novel CPGSupport Are Able To Detect Single Base Pair Mutations” Nucleic AcidsResearch 17:7187-7194.

Labels and/or quenchers can be introduced to the oligonucleotides, forexample, by using a controlled-pore glass column to introduce, e.g., thequencher (e.g., a 4-dimethylaminoazobenzene-4′-sulfonyl moiety (DABSYL).For example, the quencher can be added at the 3′ end of oligonucleotidesduring automated synthesis; a succinimidyl ester of4-(4′-dimethylaminophenylazo)benzoic acid (DABCYL) can be used when thesite of attachment is a primary amino group; and4-dimethylaminophenylazo-phenyl-4′-maleimide (DABMI) can be used whenthe site of attachment is a sulfhydryl group. Similarly, fluorescein canbe introduced into oligos, either using a fluorescein phosphoramiditethat replaces a nucleoside with fluorescein, or by using a fluoresceindT phosphoramidite that introduces a fluorescein moiety at a thymidinering via a spacer. To link a fluorescein moiety to a terminal location,iodoacetoamidofluorescein can be coupled to a sulfhydryl group.Tetrachlorofluorescein (TET) can be introduced during automatedsynthesis using a 5′-tetrachloro-fluorescein phosphoramidite. Otherreactive fluorophore derivatives and their respective sites ofattachment include the succinimidyl ester of 5-carboxyrhodamine-6G (RHD)coupled to an amino group; an iodoacetamide of tetramethylrhodaminecoupled to a sulfhydryl group; an isothiocyanate of tetramethylrhodaminecoupled to an amino group; or a sulfonylchloride of Texas red coupled toa sulfhydryl group. Labeled oligonucleotides can be purified, ifdesired, e.g., by high pressure liquid chromatography or other methods.

Similarly, signals from the labels (e.g., absorption by and/orfluorescent emission from a fluorescent label) can be detected byessentially any method known in the art. For example, multicolordetection, detection of FRET (including, e.g., time-resolved or TR-FRET,e.g., between lanthanide chelate donors and fluorescent dye acceptors;see, e.g., Journal of Biomolecular Screening (2002) 7:3-10), and thelike, are well known in the art. In brief, FRET (Fluorescence ResonanceEnergy Transfer) is a non-radiative energy transfer phenomenon in whichtwo fluorophores with overlapping emission and excitation spectra, whenin sufficiently close proximity, experience energy transfer by aresonance dipole induced dipole interaction. The phenomenon is commonlyused to study the binding of analytes such as nucleic acids, proteinsand the like. FRET is a distance dependent excited state interaction inwhich emission of one fluorophore is coupled to the excitation ofanother which is in proximity (close enough for an observable change inemissions to occur). Some excited fluorophores interact to formexcimers, which are excited state dimers that exhibit altered emissionspectra (e.g., phospholipid analogs with pyrene sn-2 acyl chains); see,e.g., Haughland (2003) Handbook of Fluorescent Probes and ResearchProducts Ninth Edition, available from Molecular Probes. Astraightforward discussion of FRET can be found in the Handbook and thereferences cited therein.

As another example, fluorescence polarization can be used. Briefly, inthe performance of such fluorescent binding assays, a typically small,fluorescently labeled molecule, e.g., a ligand, antigen, etc., having arelatively fast rotational correlation time, is used to bind to a muchlarger molecule, e.g., a receptor protein, antibody etc., which has amuch slower rotational correlation time. The binding of the smalllabeled molecule to the larger molecule significantly increases therotational correlation time (decreases the amount of rotation) of thelabeled species, namely the labeled complex over that of the freeunbound labeled molecule. This has a corresponding effect on the levelof polarization that is detectable. Specifically, the labeled complexpresents much higher fluorescence polarization than the unbound, labeledmolecule.

As those skilled in the art will recognize, any of the lipid compoundsand preparations containing the lipid compounds (including the lipid andcontrast agent preparations), may be lyophilized for storage, andreconstituted in, for example, an aqueous medium (such as sterile wateror phosphate buffered saline), with the aid of vigorous agitation. Inorder to prevent agglutination or fusion of the lipids as a result oflyophilization, it may be useful to include additives in the formulationto prevent such fusion or agglutination. Additives which may be usefulinclude sorbitol, mannitol, sodium chloride, glucose, trehalose,polyvinylpyrrolidone and polyethyleneglycol (such as PEG 400). These andother additives are described in the literature, such as in the U.S.Pharmacopeia, USP XXII, NF XVII, The United States Pharmacopeia, TheNational Formulary, United States Pharmacopeial Convention Inc., 12601Twinbrook Parkway, Rockville, Md. 20852, the disclosures of which arehereby incorporated herein by reference in their entirety. Lyophilizedpreparations generally have the advantage of greater shelf life.

The contrast agent of the invention may further, if desired, comprise asuspending agent. Preferable suspending agents include polyethyleneglycol, lactose, mannitol, sorbitol, ethyl alcohol, glycerin, lecithin,polyoxyethylene sorbitan monoleate, sorbitan monoleate and albumin. Asthose skilled in the art would recognize, various sugars and otherpolymers may also be employed, such as polyethylene,polyvinylpyrrolidone, propylene glycol, and polyoxyethylene. The amountof paramagnetic acylated MR contrast agent, e.g., Mn-DDP-EDTA, may varyfrom about 1 to 75 percent by weight of the total ingredients used toformulate the paramagnetic MR contrast agent emulsion.

The present invention is useful in imaging a patient generally, and/orin specifically diagnosing the presence of diseased tissue in a patient.The imaging process of the present invention may be carried out byadministering a contrast medium of the invention to a patient, and thenscanning the patient using magnetic resonance imaging to obtain visibleimages of an internal region of a patient and/or of any diseased tissuein that region. By region of a patient, it is meant the whole patient,or a particular area or portion of the patient.

Any of the various types of magnetic resonance imaging devices can beemployed in the practice of the invention, the particular type or modelof the device not being critical to the method of the invention. Themagnetic resonance imaging techniques which are employed areconventional and are described, for example, in Kean, D. M., and M. A.Smith, Magnetic Resonance Imaging: Principles and Applications (Williamsand Wilkins, Baltimore 1986), the disclosures of which are herebyincorporated herein by reference in their entirety. Contemplatedmagnetic resonance imaging techniques include, but are not limited to,nuclear magnetic resonance (NMR), NMR spectroscopy, and electronic spinresonance (ESR). The preferred imaging modality is NMR.

As one skilled in the art would recognize, administration of thecontrast agent to the patient may be carried out in various fashions,such as intravascularly, orally, rectally, etc., using a variety ofdosage forms. Preferably, administration is by intravascularly. Theuseful dosage to be administered and the particular mode ofadministration will vary depending upon the age, weight and theparticular animal and region thereof to be scanned, and the particularcontrast agent of the invention to be employed. Typically, dosage isinitiated at lower levels and increased until the desired contrastenhancement is achieved. By way of general guidance, typically betweenabout 0.1 mg and about 1 g of the liposoluble compound of the presentinvention, and between about 1 and about 50 micromoles of paramagneticion, each per kilogram of patient body weight, is administered, althoughhigher and lower amounts can be employed. Similarly, by way of generalguidance, where lipids or suspending agents are used in the formulation,generally between about 0.5 and about 50 percent by weight of the entireformulation of each may be employed, although higher and lower amountsmay also be used.

In carrying out the method of the present invention, the contrast agentmay be used alone, or in combination with other diagnostic, therapeuticor other agents. Such other agents include excipients such as flavoringor coloring materials.

In one embodiment, the method is particularly useful in a humansuspected of having a proliferation of a cellular mass. It can also beused with other imaging techniques and devices, as described herein.Imaging can begin pre-administration of drug using a similar compositionto determine the best liposome size or after injection to follow thebiodistribution of the liposomes carrying drugs. Typically, thecomposition is injected into a vessel of a human. Imaging comprisesimaging at least 10 hours post injection of said composition or sooner.The composition can be administered using a device selected from thegroup consisting of an intravenous syringe injection, a catheter, anintravenous drip and an intraperitoneal syringe injection. A lipiddosage range can be established using known methods and can include adose of 0.10 to 0.50 millimoles of lipid per kilogram of body weight.

Specific delivery of liposomes to a target tissue such as aproliferating cell mass, neoplastic tissue, inflammatory tissue,inflamed tissue, and infected tissue can be achieved by selecting aliposome size appropriate for delivering a therapeutic agent to saidtarget tissue. For example, liposomes with a mean diameter of 180 nm maynot accumulate in a solid tumor; preferably liposomes with a meandiameter of 140 nm accumulate in the periphery of the same solid tumor,and preferably liposomes with a mean diameter of 110 nm accumulate inthe peripheral and central portions of that solid tumor.

In another embodiment of the invention, liposome preparations ofdifferent sizes carrying imaging agents can be used to probe capillarypermeability and pore size in vivo. This information can be used todetermine the optimal particle size of liposomes carrying therapeuticagents for treatment of a particular type of disease in a fewexperiments (e.g. 2-3). Since tumors are biologically heterogeneous andeven the same tumor type may behave differently between differentpatients, this information can be very useful for tailoring liposomesize and for the most advantageous preparation for treatment of aparticular type of disease such as cancer or inflammatory tissue. Inanother embodiment, the specificity of delivery of liposomes to a targettissue may be further enhanced by labelling the liposomes withantibodies (e.g. therapeutic agents) or other tissue markers. In anotherembodiment, antibody labelling can be used to achieve or enhanceintracellular delivery of the therapeutic agent.

In one embodiment, the invention provides for a method of imagingcomprising

-   -   a) administering to a mammal need thereof a composition,        comprising:    -   i) a sufficient amount of imaging agent,    -   ii) a liposome comprising a bilayer, a fusigenic protein or        polypeptide and an interior volume, wherein said liposome is in        an amount sufficient to permit delivery of said liposome to a        tissue, and said liposome carries the Imaging agent,    -   b) imaging a tissue of the mammal.

In another embodiment, the composition used in the imaging methodfurther comprises a therapeutic agent in an therapeutic amount, whereinsaid liposome carries said therapeutic agent.

The present invention also provides for methods of drug delivery, drugdelivery monitoring, tumor killing, tumor regression, tumor growthmonitoring and drug dosing based on delivery in a mammal. The drugdelivery method can comprise:

-   -   a) administering to a mammal need thereof a composition,        comprising:    -   i) a paramagnetic chelate with a paramagnetic ion, said        paramagnetic chelate is in an amount sufficient to enhance NMR        imaging,    -   ii) a liposome comprising a bilayer, a fusigenic protein or        polypeptide and an interior volume, wherein said liposome is in        an amount sufficient to permit delivery of said liposome to a        tissue, and said liposome carries said paramagnetic chelate,    -   b) MNR imaging a tissue of said mammal.

In another embodiment, the composition used in the imaging methodfurther comprises a therapeutic agent in an therapeutic amount, whereinsaid liposome carries said therapeutic agent.

Preferably, the imaging is quantitative and amount of said liposomedelivered to said tissue can be estimated and the amount of selectivelydelivered drug calculated. These methods can be combined with methods ofmonitoring tissue mass to evaluate the therapeutic effectiveness of thedrug delivery method and the drug. For instance, determining the volumeof the tissue in order to monitor tissue volume, to indicate tissueproliferation, or to monitor a reduction in tissue mass can beaccomplished. Such methods may also be used to determine the optimaldelivery regime to a particular pathologic tissue in a particularpatient.

In the case of diagnostic applications, such as ultrasound and CT,energy, such as ultrasonic energy, is applied to at least a portion ofthe patient to image the target tissue. A visible image of an internalregion of the patient is then obtained, such that the presence orabsence of diseased tissue can be ascertained.

Ultrasound can be used for both diagnostic and therapeutic purposes. Indiagnostic ultrasound, ultrasound waves or a train of pulses ofultrasound may be applied with a transducer. The ultrasound is generallypulsed rather than continuous, although it may be continuous, ifdesired. Thus, diagnostic ultrasound generally involves the applicationof a pulse of echoes, after which, during a listening period, theultrasound transducer receives reflected signals. Harmonics,ultraharmonics or subharmonics may be used. The second harmonic mode maybe beneficially employed, in which the 2× frequency is received, where xis the incidental frequency. This may serve to decrease the signal fromthe background material and enhance the signal from the transducer usingthe targeted contrast media of the present invention which may betargeted to the desired site, for example, blood clots. Other harmonicssignals, such as odd harmonics signals, for example, 3x or 5x, would besimilarly received using this method. Subharmonic signals, for example,x/2 and x/3, may also be received and processed so as to form an image.

In addition to the pulsed method, continuous wave ultrasound, forexample, Power Doppler, may be applied. This may be particularly usefulwhere rigid vesicles, for example, vesicles formulated from polymethylmethacrylate, are employed. In this case, the relatively higher energyof the Power Doppler may be made to resonate the vesicles and therebypromote their rupture. This can create acoustic emissions which may bein the subharmonic or ultraharmonic range or, in some cases, in the samefrequency as the applied ultrasound. It is contemplated that there willbe a spectrum of acoustic signatures released in this process and thetransducer so employed may receive the acoustic emissions to detect, forexample, the presence of a clot. In addition, the process of vesiclerupture may be employed to transfer kinetic energy to the surface, forexample of a clot to promote clot lysis. Thus, therapeutic thrombolysismay be achieved during a combination of diagnostic and therapeuticultrasound. Spectral Doppler may also be employed. In general, thelevels of energy from diagnostic ultrasound are insufficient to promotethe rupture of vesicles and to facilitate release and cellular uptake ofthe bioactive agents. As noted above, diagnostic ultrasound may involvethe application of one or more pulses of sound. Pauses between pulsespermits the reflected sonic signals to be received and analyzed. Thelimited number of pulses used in diagnostic ultrasound limits theeffective energy which is delivered to the tissue that is being studied.

Higher energy ultrasound, for example, ultrasound which is generated bytherapeutic ultrasound equipment, is generally capable of causingrupture of the vesicle species. In general, devices for therapeuticultrasound employ from about 10 to about 100% duty cycles, depending onthe area of tissue to be treated with the ultrasound. Areas of the bodywhich are generally characterized by larger amounts of muscle mass, forexample, backs and thighs, as well as highly vascularized tissues, suchas heart tissue, may require a larger duty cycle, for example, up toabout 100%.

In therapeutic ultrasound, continuous wave ultrasound is used to deliverhigher energy levels. For the rupture of vesicles, continuous waveultrasound is preferred, although the sound energy may be pulsed also.If pulsed sound energy is used, the sound will generally be pulsed inecho train lengths of from about 8 to about 20 or more pulses at a time.Preferably, the echo train lengths are about 20 pulses at a time. Inaddition, the frequency of the sound used may vary from about 0.025 toabout 100 megahertz (MHz). In general, frequency for therapeuticultrasound preferably ranges between about 0.75 and about 3 MHz, withfrom about 1 and about 2 MHz being more preferred. In addition, energylevels may vary from about 0.5 Watt (W) per square centimeter (cm·2) toabout 5.0 W/cm·2, with energy levels of from about 0.5 to about 2.5W/cm·2 being preferred. Energy levels for therapeutic ultrasoundinvolving hyperthermia are generally from about 5 W/cm·2 to about 50W/cm·2. For very small vesicles, for example, vesicles having a diameterof less than about 0.5 μm, higher frequencies of sound are generallypreferred. This is because smaller vesicles are capable of absorbingsonic energy more effectively at higher frequencies of sound. When veryhigh frequencies are used, for example, greater than about 10 MHz, thesonic energy will generally penetrate fluids and tissues to a limiteddepth only. Thus, external application of the sonic energy may besuitable for skin and other superficial tissues. However, it isgenerally necessary for deep structures to focus the ultrasonic energyso that it is preferentially directed within a focal zone.Alternatively, the ultrasonic energy may be applied via interstitialprobes, intravascular ultrasound catheters or endoluminal catheters.Such probes or catheters may be used, for example, in the esophagus forthe diagnosis and/or treatment of esophageal carcinoma. In addition tothe therapeutic uses discussed above, the present compositions can beemployed in connection with esophageal carcinoma or in the coronaryarteries for the treatment of atherosclerosis, as well as thetherapeutic uses described, for example, in U.S. Pat. No. 5,149,319, thedisclosures of which are hereby incorporated herein by reference, intheir entirety.

A therapeutic ultrasound device may be used which employs twofrequencies of ultrasound. The first frequency may be x, and the secondfrequency may be 2x. In preferred form, the device would be designedsuch that the focal zones of the first and second frequencies convergeto a single focal zone. The focal zone of the device may then bedirected to the targeted compositions, for example, targeted vesiclecompositions, within the targeted tissue. This ultrasound device mayprovide second harmonic therapy with simultaneous application of the xand 2x frequencies of ultrasound energy. It is contemplated that, in thecase of ultrasound involving vesicles, this second harmonic therapy mayprovide improved rupturing of vesicles as compared to ultrasound energyinvolving a single frequency. Also, it is contemplated that thepreferred frequency range may reside within the fundamental harmonicfrequencies of the vesicles. Lower energy may also be used with thisdevice. An ultrasound device which may be employed in connection withthe aforementioned second harmonic therapy is described, for example, inKawabata, K. et al., Ultrasonics Sonochemistry, Vol. 3, pp. 1-5 (1996),the disclosures of which are hereby incorporated herein by reference, intheir entirety.

In connection with methods involving ultrasonic imaging, particularly inembodiments involving vesicles, diagnostic ultrasound imaging may becarried out simultaneously with the application of therapeuticultrasonic waves so as to rupture the vesicles for purposes, such as,for example, enhanced cavitation or the targeted release of a bioactiveagent combined with the vesicles. The method comprises the steps of (i)administering to the patient a quantity of vesicles; (ii) insonating thevesicles in a region of the patient with therapeutic ultrasonic waves ata frequency and energy to cause the vesicles to rupture; and (iii)simultaneously receiving ultrasonic emissions from the insonatedvesicles at a harmonic of the frequency of the therapeutic ultrasonicwaves and generating an image of said region from the receivedultrasonic emissions. Simultaneous imaging allows an operator to monitorthe rupture of the vesicles in real time.

As one skilled in the art would recognize, once armed with the teachingsin the present disclosure, widely varying amounts of vesicles may beemployed in the practice of the methods described herein. As usedherein, the term “quantity of vesicles” is intended to encompass allsuch amounts.

Diagnostic imaging is a means to visualize internal body regions of apatient. Diagnostic imaging includes, for example, ultrasound (US),magnetic resonance imaging (MRI), nuclear magnetic resonance (NMR),computed tomography (CT), electron spin resonance (ESR); nuclearmedicine when the contrast medium includes radioactive material; andoptical imaging, particularly with a fluorescent contrast medium.Diagnostic imaging also includes promoting the rapture of the vesiclesvia the methods of the present invention. For example, ultrasound may beused to visualize the vesicles and verify the localization of thevesicles in certain tissue. In addition, ultrasound may be used topromote rapture of the vesicles once the vesicles reach the intendedtarget, including tissue and/or receptor destination, thus releasing abioactive agent and/or diagnostic agent.

In accordance with the present invention, there are provided methods ofimaging a patient generally, and/or in specifically diagnosing thepresence of diseased tissue in a patient. The imaging process of thepresent invention may be carried out by administering a contrast mediumof the invention to a patient, and then scanning the patient using, forexample, ultrasound, computed tomography, and/or magnetic resonanceimaging, to obtain visible images of an internal region of a patientand/or of any diseased tissue in that region. By region of a patient, itis meant the whole patient or a particular area or portion of thepatient.

In employing the contrast agents, they are preferably suspended inaqueous solution and the contrast medium formulated using steriletechniques. An advantage to using smaller liposomes (e.g., 200 nm andbelow in size) and micelles or emulsified lipids, as well as the simplesuspension of paramagnetic ions and liposoluble compounds, is that thecontrast agents may be filtered through 0.22 micron line filters eitherimmediately prior to administration, such as by intravenous injection,or as a terminal step in formulation of the contrast agents, to removeany potential pyrogens.

For formulating these contrast agents into stable preparations otheradditives may be employed. For example, in formulating contrast agentsfor intravenous injection, parenteral additives may be included in thepreparation. Such additives to include tonicity adjusting additives suchas dextrose and sodium chloride, to formulate an isosmotic contrastmedium. These tonicity additives are generally provided in minoramounts, such as about 0.1% to about 0.5% by weight of the totalformulation. In addition, antimicrobial additives may be included in thefinal preparation so as to avoid bacterial growth. Such antimicrobialadditives, in generally acceptable amounts, may include but are notlimited to benzalkonium chloride (typically 0.01% by weight of the totalformulation), benzyl alcohol (typically 1-2% by weight), chlorobutanol(typically 0.25-0.5% by weight), metacresol (typically 0.1-0.3% byweight), butyl p-hydroxybenzoate (typically 0.015% by weight), methylp-hydroxybenzoate (typically 0.1-0.2% by weight), propylp-hydroxybenzoate (typically 0.2% by weight), phenol (0.25-0.5% byweight) and thimerosal (typically 0.01% by weight). Additionally,antioxidants may be included in the preparation, and are particularlyuseful where the contrast agent contains unsaturated lipids. Suchantioxidants in their generally useful amounts include ascorbic acid(typically 0.01-0.5% by weight), cysteine (typically 0.1-0.5% byweight), monothioglycerol (typically 0.1-1.0% by weight), sodiumbisulfite (typically 0.1-1.0% by weight), sodium metabisulfite(typically 0.1-1.0% by weight), and tocopherols (typically 0.05-0.5% byweight). As those skilled in the art will recognize, the contrast agentsof the invention may be formulated in a variety of means to beparticularly suitable for intravascular delivery, delivery into any bodycavity, or other delivery targets.

Additional Agents

It is also contemplated to be a part of the present invention to preparemicrospheres using compositions of matter in addition to thebiocompatible lipids and polymers described above, provided that themicrospheres so prepared meet the stability and other criteria set forthherein.

Propylene glycol may be added to remove cloudiness by facilitatingdispersion or dissolution of the lipid particles. The propylene glycolmay also function as a thickening agent which improves microsphereformation and stabilization by increasing the surface tension on themicrosphere membrane or skin. It is possible that the propylene glycolfurther functions as an additional layer that coats the membrane or skinof the microsphere, thus providing additional stabilization. As examplesof such further basic or auxiliary stabilizing compounds, there areconventional surfactants which may be used, e.g., U.S. Pat. Nos.4,684,479 and 5,215,680.

Additional auxiliary and basic stabilizing compounds include such agentsas peanut oil, canola oil, olive oil, safflower oil, corn oil, or anyother oil commonly known to be ingestible which is suitable for use as astabilizing compound in accordance with the requirements andinstructions set forth in the instant specification.

In addition, compounds used to make mixed micelle systems may besuitable for use as basic or auxiliary stabilizing compounds, and theseinclude, but are not limited to: lauryltrimethylammonium bromide(dodecyl-), cetyltrimethylammonium bromide (hexadecyl-),myristyltrimethylammonium bromide (tetradecyl-),alkyldimethylbenzylammonium chloride (alkyl=C₁₂,C₁₄,C₁₆),benzyldimethyldodecylammonium bromide/chloride, benzyldimethylhexadecylammonium bromide/chloride, benzyldimethyl tetradecylammoniumbromide/chloride, cetyl-dimethylethylammonium bromide/chloride, orcetylpyridinium bromide/chloride.

It has been found that the liposomes used in the present invention maybe controlled according to size, solubility and heat stability bychoosing from among the various additional or auxiliary stabilizingagents described herein. These agents can affect these parameters of themicrospheres not only by their physical interaction with the lipidcoatings, but also by their ability to modify the viscosity and surfacetension of the surface of the liposome. Accordingly, the liposomes usedin the present invention may be favorably modified and furtherstabilized, for example, by the addition of one or more of a widevariety of (a) viscosity modifiers, including, but not limited tocarbohydrates and their phosphorylated and sulfonated derivatives; andpolyethers, preferably with molecular weight ranges between 400 and100,000; di- and trihydroxy alkanes and their polymers, preferably withmolecular weight ranges between 200 and 50,000; (b) emulsifying and/orsolubilizing agents may also be used in conjunction with the lipids toachieve desired modifications and further stabilization; such agentsinclude, but are not limited to, acacia, cholesterol, diethanolamine,glyceryl monostearate, lanolin alcohols, lecithin, mono- anddi-glycerides, mono-ethanolamine, oleic acid, oleyl alcohol, poloxamer(e.g., poloxamer 188, poloxamer 184, and poloxamer 181), polyoxyethylene50 stearate, polyoxyl 35 castor oil, polyoxyl 10 oleyl ether, polyoxyl20 cetostearyl ether, polyoxyl 40 stearate, polysorbate 20, polysorbate40, polysorbate 60, polysorbate 80, propylene glycol diacetate,propylene glycol monostearate, sodium lauryl sulfate, sodium stearate,sorbitan mono-laurate, sorbitan mono-oleate, sorbitan mono-palmitate,sorbitan monostearate, stearic acid, trolamine, and emulsifying wax; (c)suspending and/or viscosity-increasing agents that may be used with thelipids include, but are not limited to, acacia, agar, alginic acid,aluminum mono-stearate, bentonite, magma, carbomer 934P,carboxymethylcellulose, calcium and sodium and sodium 12, carrageenan,cellulose, dextran, gelatin, guar gum, locust bean gum, veegum,hydroxyethyl cellulose, hydroxypropyl methylcellulose,magnesium-aluminum-silicate, methylcellulose, pectin, polyethyleneoxide, povidone, propylene glycol alginate, silicon dioxide, sodiumalginate, tragacanth, xanthum gum, alpha-d-gluconolactone, glycerol andmannitol; (d) synthetic suspending agents may also be utilized such aspolyethyleneglycol (PEG), polyvinylpyrrolidone (PVP), polyvinylalcohol(PVA), polypropylene glycol, and polysorbate; and (e) tonicity raisingagents may be included; such agents include but are not limited tosorbitol, propyleneglycol and glycerol.

The diluents which can be employed to create an aqueous environmentinclude, but are not limited to water, either deionized or containingany number of dissolved salts, etc., which will not interfere withcreation and maintenance of the stabilized microspheres or their use asMRI contrast agents; and normal saline and physiological saline.

Although this invention has been described in connection with its mostpreferred embodiment, additional embodiments are within the scope andspirit of the claimed invention. The preferred device of this inventionis intended merely to illustrate the invention, and not limit the scopeof the invention as it is defined in the claims that follow.

EXPERIMENTAL EXAMPLES Example 1: Saposin C and Liposome Preparation andDelivery In Vitro and in Vivo

Materials—

The following materials are from commercial sources: mouse laminin, P/S,fetal bovine serum, and DMEM (Gibco BRL, Gaithersborg, MD); Neurobasalmedium with B27 supplement (Life Technologies); restrictionendonucleases (New England Biolabs, Beverly, Mass.); pET21a(+) DNAvector, E. Coli host strain [BL21(DE3)], and His•Bind resin (Novagen,Medison, WI); monoclonal anti-His antibody conjugated with AlexaFluor488 (QIAGEN, Valencia, Calif.); fluorescein-conjugated goatanti-rabbit and rhodamine-conjugated sheep anti-mouse antibodies(ICN/CAPPEL, Aurora, Ohio); antifade reagent (Ventana Medical Systems,Tucson, Ariz.); C₄ reverse-phase HPLC column (Alltech Association Inc.,Deerfield, Ill.); DOPS and1,2-Dioleoyl-sn-Glycero-3-Phospho-L-Serine-N-(7-nitro-2-1,3-benzoxadiazol-4-yl)(NBD-DOPS) as stock solutions in chloroform (Avanti Polar Lipids,Alabaster, Ala.); polyethylenimine and papain (Sigma, St. Louis, Mo.).Anionic lipids are sodium salts. All other chemicals are reagent gradeor better.

Fibroblast Cell Cultures—

Human and mouse primary fibroblasts are used for all experiments andestablished with standard procedures in this laboratory.¹⁵ Mouseprosaposin deficient fibroblasts from PSAP−/− mice. All the cells arecultured in DMEM/FBS (10%) media at 37° C. in monolayer for next use.

Primary Cortical Neuron Cultures—

Cortical neurons are cultured in serum-free Neuroblasal medium with B27supplements as described by Whitmarsh et al.⁴⁴ Take out E16 mouseembryos, cut the head and place them into ice-cold Ca/Mg-free HankBalance Salt Solution (HBSS) with papain (1 mg/ml). The brain isdissected out and place scalpel along the dorsal midline between the twocerebral hemispheres but slightly deviate towards to the side as it cut.This will give a clean cerebral cortex. Peel out the meninges gentlywithout touching the medial side of the cortex where the hippocampus islocated. Cut out the cortex with curved fine surgical scissors andcollect them in ice-cold HBSS. The cortical tissue is replaced in papainHBSS solution for 15-20 minutes at room temperature to soften up thetissues. Transfer them to papain-inhibitor solution for another 5minutes at room temperature and finally back to 2 ml ice-cold HBSS.Fisherbran 12-546 (18CIR-2) coverglasses in 12-well plate are coatedwith PEI containing laminin overnight. The isolated cortical tissues arecultured on the PEI coated coverglass in the plate with Neurobasal/B27medium. Kainate treatment is performed by addition of the drug to themedium.

Saposin C and Liposomes Preparation

Recombinant saposin C is routinely produced using IPTG-inducing pETsystem in E. coli cells in our laboratory.¹¹ All expressed proteinscontained a His-tag, and are purified on a nickel column and with C4reverse phase HPLC chromatography using a linear (0-100%) gradient ofacetonitrile in 0.1% trifluoroacetic acid. The major protein peak iscollected and lyophilized. The protein concentrations are determined aspreviously described by Qi et al.¹¹

DOPS lipids (16.2 μg) in chloroform are dried under N₂ and vacuum toform a lipid film. Saposin C (79 μg) is added into the lipid film, andare suspended in 50 μl of 0.1 M citric acid/0.2 M phosphate (pH 4.7).Additional medium or PBS is added. Large unilamellar vesicles (LUV) areprepared by bath sonications.¹⁴ Liposome size is measured by photoncorrelation spectroscopy with a N4+ submicron particle size analyzer(Coulter, Miami, Fla.). The sizes of the populations of LUV areevaluated using the N4+ sub-micron particle size analyzer and aredispersed with an average diameter 250±100 nm.

Delivery of Saposin C-DOPS Proteoliposomes In Vitro and In Vivo

Cells (10⁵) are grown in DMEM medium for 48 h in a 8 wells chamber slidewith coverglass (Lab-Tek II, Nalge Nunc International). Saposin C-DOPScomplex in the medium is added into cell cultures. After incubated at37° C. for 48 h, the cells are washed with PBS twice, and fixed with 2%paraformaldehyde for immunofluorescence assay. For in vivo study, theproteoliposomes in PBS are injected into mice through tail veins. Mousebrain tissues are collected at 48 h after administration of theprotein-lipid complex for immunofluorescence assay.

Histopathology and Immunofluorescence

Mice brain tissues are fixed and snap frozen in 10% formalin prior to beprocessed. The paraffin sections are stained with hematoxylin and eosin(H&E), and analyzed under a light microscopy.

Immunofluorescence staining is done as described with minormodification.¹⁵ Cultured cells (1×10⁵) in a dish with coverslips arewashed with PBS and fixed with 2% paraformaldehyde for 10 min at roomtemperature. After treating with 0.1% Triton X-100 in PBS, the samplesare incubated with each respective primary antiserum (for 2 h) andfluorescence-conjugated secondary antibody (for 1 h) at 37° C. Thedilutions of primary and secondary antibodies are 1:30 and 1:60,respectively. Mouse brain tissue sections in 4% paraformaldehyde areincubated with a block solution contains 5% mouse serum prior toaddition of primary anti-His antibody. Rhodamine-conjugated anti-mouseantibody is used as secondary antibody for detection. Antifade is addedon the section to prevent the fluorescence quenching. Fluorescencesignals are detected by a confocal microscopy (LSM510, Zeiss) or afluorescence microscopy (Zeiss Axioskop).

Example 2: Synthesis of Liposomes Using Acidic Long-Chain Lipids,Neutral Long-Chain Lipids and Neutral Short-Chain Lipids Materials andMethods

All the phospholipids DOPS, DPPC and DHPC are purchased in powder formfrom Avanti polar lipids and used without further purification. Fordynamic light scattering (DLS) measurements, the molar ratio of DOPS toDPPC in the mixtures ranges from about 10 to about 1 with([DPPC]+[DOPS])/DHPC=about 4 for all the samples. The lipid mixtures aredissolved in filtered ultra-pure H₂O (Millipore EASYpure UV) at a totallipid concentration of 10 wt. % using a combination of vortexing andtemperature cycling, between 50 and 4° C. The homogenized 10 wt. %solutions are then progressively diluted into 5, 2, 1, 0.5 and 0.1 wt. %with filtered H₂O.

Prior to DLS, stock lipid samples are diluted 5, 50 and 200 fold and areanalyzed using an N4⁺ particle sizer (Coulter, Miami, Fla.). It isdetermined that diluting the system had no effect on size determination.In the case of SANS experiments, the same sample preparation protocol isapplied to the [DOPS]/[DPPC]=10 sample except that, D₂O (99.9%, ChalkRiver Laboratories, Chalk River, ON) instead of H₂O is used to obtain asample having a total lipid concentration of 0.5 wt. %. The 0.5 wt. %solution is then further diluted into 0.1 and 0.05 wt. % mixtures usingan acidic buffer composed of equal-volumes of 0.1N sodium acetate (NaAc)and 0.1N acetic acid (HAc). The resultant solution had a pH value of4.78±0.02 in D₂O, and the buffer's pH is stable over 12 times dilutionwith D₂O.

In the case of SANS experiments, the same sample preparation procedureis applied to the [DOPS]/[DPPC]=10 sample except that D₂O (99.9%, ChalkRiver Lab.) is in replacement of filtered H₂O until the total lipidconcentration is 0.5 wt. %. The 0.5 wt. % solution is then diluted into0.1 and 0.05 wt. % with an acidic buffer composed of equal-volumemixture of 0.1N sodium acetate (NaAc) and 0.1N acetic acid (HAc)solution yielding a pH value of (4.78±0.02) in D₂O. The pH value ofbuffer is stable over 12 times of dilution with D₂O.

SapC is overexpressed in E. coli cells by using IPTG-inducing pET system(26). Expressed proteins with a His-tag are eluted from nickel columns.After dialysis, the proteins are further purified by HPLC chromatographyas follows: The C4 reverse phase column is equilibrated with 0.1%trifluoroacetic acid (TFA) for 10 minutes, and then, the proteins areeluted in a linear (0-100%) gradient of 0.1% TFA in acetonitrile over 60minutes. The major protein peak is collected and lyophilized. Theprotein concentrations are determined as previously described. Qi et al,1994.

H1 (YCEVCEFLVKEVTKLID) and H2 (EKEILDAFDKMCSKLPK) peptides aresynthesized by SynPep Corp. (California, USA) and dissolved in D₂O at aconcentration of 1.5 mg/mL. The 0.1 wt. % lipid solution with[DOPS]/[DPPC]=10 and ([DPPC]+[DOPS])/DHPC=4 is then individually addedwith the two peptide solutions (1.5 mg/mL) at a volume ratio of about12:1 and the SapC solution with a volume ratio of about 12:1 to yieldthe final peptide (or SapC) concentration of 62.5 μM, which is greaterthan the SapC concentration needed to induce membrane destabilization.(Wang, et al., 2003).

The SANS experiment is conducted at one of the 30 m SANS instruments,NG7, located at National Institute of Standards and Technology (NIST)Center for Neutron Research (NCNR, Gaithersburg, Md., USA). Awavelength, λ, of 8.09 Å and neutron focusing lens in combination of along sample-to-detector distance (SDD) of 15.3 m are used to procuresmaller values of scattering vector, q=4π/λ·sin(θ/2), where θ is thescattering angle. The other two SDD of 5 and 1 m are also employed tocover a whole q range from 0.002 to 0.35 Å⁻¹. The raw 2-D data are thencorrected by the detector sensitivity, background, empty cell scatteringand transmission of the sample, and are then circularly averaged aroundthe beam center to yield 1-D data. The 1-D data are put on the absolutescale according to the flux of the direct beam. The incoherent plateauis determined averaging the intensity of the last 10˜20 data points andsubtracted from the reduced data.

Liposome size is measured by photon correlation spectroscopy with a N4+sub-micron particle size analyzer (Coulter, Miami, Fla.) as described(14, 15). The sizes of the populations of LUV are evaluated using theN4+ sub-micron particle size analyzer and are polydispersed with anaverage diameter between 20-800 nm. The data for liposome sizeestimation is acquired at a 90° angle and processed using sizedistribution process (SDP) analysis with a fair autocorrelationfunction. The size is presented with a major fraction of vesicles by SDPdetermination. Statistical significance is estimated with ANOVAanalysis. Error bars denote standard deviation.

Transmission electron microscopy (TEM) images are taken with a HitachiTEM (H-7600, HITACHI, Japan). A droplet of each sample is placed on anickel grid coated with a support formvar film (200 mesh, a thicknessrange from 30 to 75 nm, Electron Microscopy Sciences, PA). The grid isplaced on the filter paper at room temperature for 2 h prior to TEManalysis. The TEM is operated at an acceleration voltage of 80 kV. Theimaging background is optimized at high magnification while the area ofinterest is located at low magnification (50-1,000 ×). A single vesicleis focused on using up to 50,000× magnification. Contrast and brightnessare manually adjusted until a “sharp” image is obtained and imagingbackground is optimized at high magnification. TEM micrographs are takenusing a dual AMT CCD digital camera (2K×2K, 16 bit) with appropriateimage acquisition software.

With respect to the embodiment using short-chain lipids, the generallyaccepted model for the kinetics of forming low-polydispersity ULV isdescribed as follows:

Initially, the discoidal micelle precursor starts to form with theshort-chain lipid coating at the rim and long-chain lipids at the planarbilayered surface of the disks to minimize the curvature energy at therim. Either dilution or temperature elevation causes the loss of theshort-chain lipid at the rim to the bilayer or solution, resulting in anincrease of line tension and consequently coalescence between disks toform larger disks. As the increase of line tension overwhelms thecoalescence of the nearby discoidal micelles, the contour length of therim has to decrease, causing the bilayer to fold into a spherical shellwith an opening, whose rim is covered by the short-chain lipid.Eventually, the opening can close up with the disappearance of theshort-lipid around the rim, resulting in the morphology of vesicles.

Example 3: MR Detection of Tumor Cells Labeled with USPIO Using DOPSLiposomes

To prepare the liposomes containing MR detectable labels such as USPIO,the following method is used. Sonication of dextran coated USPIOparticles in aqueous solution with DOPS does not yield sufficientencapsulation in the liposomes. In order to increase USPIO content inliposomes, a chemical coupling method as described by Bogdanov et al,Trapping of dextran-coated colloids in liposomes by transient binding toaminophospholipid: preparation of ferrosomes. Biochim Biophys Acta,1994. 1193(1): p. 212-8 is used with minor modifications. Briefly, thedextran coating on the USPIO particles is oxidized to generate aldehydegroups. Aldehydes form a covalent Schiff bond at high pH with amines ofDOPS. Liposomes obtained have a mean size of 150 nm as confirmed by N4+Particle Sizer (Beckman Coulter, CA) analysis. The liposome solution isdialyzed against a low pH solution to detach USPIO bound to the externallayer of the liposomes. Unencapsulated USPIO are removed by affinitychromatography using a Con-A Sepharose 4B column (Amersham BiosciencesCorp., NJ). The USPIO-DOPS liposome structure is confirmed byconventional electron microscopy. A standard R2 relaxivity curvegenerated using known quatities of free USPIO and DOPS liposome mixturesis used to estimate the iron concentration in the DOPS liposomes. Amaximum content of 32 μg Fe/ml is achieved using 1 mM DOPSconcentration. Four samples of neuroblastoma cells are prepared withapproximately 10,000 cells per group. The first and second samples areincubated with 100 uM and 300 μM USPIO-DOPS liposome preparation ingrowth medium respectively. The third sample contained cells with noUSPIO or liposomes. After incubation for 36 hours, the cells are washed4 times, trypsinized and fixed in a mixture of 0.5% agarose solution andgrowth medium (1:1) in 4 ml glass vials.

High resolution MR imaging of the cells is performed using a 7T BrukerBiospec scanner using gradient echo methods optimized for T2 weighting.A 3D FLASH imaging sequence with TR/TE/0 of 200 ms/35 ms/10° and a320×320×64 matrix is used for a 3.2 cm×3.2 cm×0.64 cm FOV resulting inan isotropic 100 um resolution.

The MR images indicates uptake of USPIO particles by cells in samples 1and 2, with sample 2 showing an increased uptake corresponding to thehigher concentration of USPIO-DOPS liposomes. A much lower number ofcells is detected in sample containing cells with liposome-USPIOsolution prepared by sonication. An estimate of the number of cellsdetected in each vial is obtained using a post-processing algorithmwritten in IDL. The number of cells detected invial 2 is approximately1.4 times compared to vial 1. The average contrast-to-noise ratio (CNR)between the gel and hypo-intensity regions representing cells is 20.15,SD 11.

Example 4: Preparation of SapC-DOPS Proteoliposomes

Protonation of SapC is used to promote the bind of SapC and DOPSmembranes. First, SapC is protonated by dissolving in an aliquot acidicbuffer (pH 5, 20 μl), then diluted with PBS or neutral buffer (pH 7)into 1 ml final volume. Alternatively, Brønsted acid (such as TFE,chloroform, methonal, etc.) can be used to dissolve SapC with DOPSlipids. These Bronsted acids have been reported to be a good H-bonddonor and to have a protonation effect on the proteins (1). Thesesolvents can be evaporated to dry under N2 gas or a vaccume system.Suitable plecable (such as PBS) is added to form SapC-DOPSproteoliposomes. This procedure is to avoid the DOPS liposome fusioninduced by SapC at acidic pH. The proteoliposomes prepared by thisapproach are in a mono-disperse form with an average size at 200 nm.

Example 5: Temperature Control Leakage of SapC-DOPS Proteoliposomes

SapC-DOPS proteoliposomes are designed with various lipid compositionsto be sensitive to the temperature for leakage of the contentsencapsulated into the liposomes.

Example 6: Characterization of Liposomes/Saposin C

To elucidate the temporal and spatial interaction of saposins withliposomal membranes, the Inventor has focused efforts on the developmentof intrinsic (Trp) and/or extrinsic (NBD, pyrene, etc.) fluorescencedetermination methods. These approaches include maximal emissionspectrum shift, fluorescence resonance energy transfer, fluorescencestopped-flow analysis, flow-analysis of fluorescentbead-saposin-liposome complexes, and fluorescence microscopy. Inaddition, circular dichroism (CD) is used to evaluate relative secondarystructure changes from lipid-free to lipid-bound saposins. Analyses ofthe initial results evolved into the proposed hypothesis.

Summarized below are the studies related to the expression,purification, functional analysis, mutagenesis, as well as fluorescenceanalyses of saposin-phospholipid interaction and membrane fusion.

I. Purification and Characterization of Natural and Recombinant Saposins

a) Expression of Saposins from Prokaryotic Systems

Although natural saposins have been isolated and characterized, it isimportant to establish a recombinant expression system to provide anaccessible source of large amounts of normal, mutated and Trp-labeledsaposins for the proposed investigations. A prokaryotic system isdeveloped, based on the following: 1) Saposins have at least oneoccupied N-glycosylation site, but, for saposins B and C, occupancy ofthese sites are not needed for function. 2) Expression of proteins ineukaryotic systems is labor and resource intensive, and slow. Incomparison, prokaryotic systems are rapid and give high yields ofwild-type and mutant proteins. And 3) The proteins can be labeled withTip residues as intrinsic fluorescence probes, since wild-type A is theonly saposin that contains a natural Trp (37W).

b) Production of Active Saposins in E. Coli

Functional saposins were overexpressed in BL21(DE3) cells using a pET21a series vector. Following IPTG induction at 37° C. or 30° C., largeamounts of saposins containing His⋅Tag were found in the solublefraction of the disrupted cells. These were conveniently purified toelectrophoretic homogeneity on nickel-loaded columns. Alternatively,saposins without His⋅Tag were generated by introducing a stop codonafter protein coding region, and then purified using immuno-affinitycolumns with T7-taq monoclonal antibody. The purified recombinantsaposin C shows excellent activation of acid β-glucosidase and otherbiologic properties. Circular dichroism spectra, light scattering, andES-MS analyses were used to evaluate the physical properties of thepurified saposins, such as aggregation status and molecular weight.Trp-saposins without His⋅Tag were also generated for necessary controlexperiments. The functional integrity of recombinant saposin C isdetermined using delipidated and homogenous acid β-glucosidase in aliposomal reconstitution system and in neuritogenic assays. Recombinantsaposin B function is determined using a sulfatide binding assay. The invitro function of recombinant saposins B and C are similar to thenatural or deglycosylated saposins.

II. Functional Conformations of Saposins Induced by Phospholipids

To determine the specificity of saposin C-phospholipid interaction, aliposomal system is developed using CD, fluorescence emission shifts,and fluorescence quenching methods. Mutated saposins C's, produced tocontain individual Trp (W), are termed saposin C(0W), (S37W), and (81W).These Trp-labeled saposin C's are as follows: saposin C(0W) has a Trppreceding the first NH₂-terminal amino acid of mature saposin C, saposinC(S37W) has a Trp at residue 37 (i.e., in the middle), and saposinC(81W) has a Trp after the last COOH-terminal amino acid. Thesesubstitutions had no effect on the activation properties or CD spectraof saposin C.

a) CD Spectra

Using CD spectroscopy, relative secondary structural changes ofrecombinant saposins are induced by membrane binding. The relativesecondary structural changes of saposins obtained from the acidic,unsaturated phosphatidylserine (PS)/saposin C complexes and the neutralphosphatidylcholine (PC)/saposin B complexes are similar and result in adecrease the β-strand and an increase the α-helix content (Table 4).

TABLE 4 Table 4. Circular Dichroism (195-250 nm) Analyses of Saposinswith Various Phospholipids II. Saposin % α % β % T % R C Only 29.9 41.70.0 28.4 C + Phosphatidylserine (18:0,0) 30.1 40.4 1.4 28.1 C +Phosphatidylcholine (18:1,1) 30.6 41.0 0.0 28.4 C + Phosphatidylserine(18:1,1) 49.8 3.9 14.0 32.4 B only 43.7 36.6 0.0 19.7 B +Phosphatidylserine (18:1,1) 43.8 38.8 0.0 17.9 B + Phosphatidylcholine(18:1,1) 68.2 24.2 5.3 2.3 A only 44.0 31.9 0.0 24.1 A +Phosphatidylserine (18:1,1) 39.3 34.9 0.5 25.4

No changes are observed with saposin A and B in the PS (18:1,1)complexes. These results indicate that saposin A and B have a differentmembrane interaction from that of saposins C. The CD data are collectedon a Jasco 710 instrument, and deconvoluted using Yang's method (seeChang, C. T., Wu, C. S., and Yang, J. T. Anal. Biochem (1978) 91,13-31).

b) Fluorescence Emission Spectra

Emission spectra of proteins shift when the tryptophanyl environmentschange polarity. The fluorescence spectra of saposins A(0W), A(37W),A(81W), C(0W), and C(81W) obtained upon addition of brainphosphatidylserine (BPS) liposomes, showed blue-shifts (Table 5).

TABLE 5 Table 5. Fluorescence Emission Maxima of Trp-saposins in theAbsence and Presence of Brain phosphatidylserine (BPS) Emission Maxima(EM, nm) Saposins −BPS +BPS EM Shifts C(0W) 339 333 Blue C(S37W) 351 351No C(S37W, Q48N) 345 339 Blue C(S37W, Q48A/E49A) 338 329 Blue C(81W) 339323 Blue A(0W) 345 333 Blue A(37W) 351 338 Blue A(37W, G64E) 344 358 RedA(37W, 339 350 Red K63L/G64E/M65V) A(81W) 345 336 Blue

Experiments conditions: pH 4.7, protein:lipid=1:20 to 40. No differencesare observed at 22 or 37° C.

The blue-shifts suggest interaction of saposins with lipids duringcomplex formation. However, saposin C (S37W) shows no shift in thepresence of BPS. This implies that the NH₂-(0W) and COOH-(81W) terminiof saposin C enter the membrane whereas the middle of the sequence doesnot. With saposin A, the reverse is true with the middle of the sequence(37W) in the membrane. This means that saposin A-membrane associationsare quite different from those of the saposin C. These results areconsistent with the CD analysis. Maximal emission wavelength changes arenot observed with saposin As or Cs in the presence of neutral EPC norwith PS containing saturated fatty acid chains.

1. Temporal and Spatial Interaction of Saposins and PhospholipidMembranes

To investigate temporal and spatial interactions of saposins andliposomal membrane, fluorescence stopped-flow and quenching approachesare used with Trp as the intrinsic fluorescence probe of the saposins.These experiments allow identification of regional interactions betweensaposins and lipid bilayers, and also the kinetics of their binding.

Temporal Interactions

Fluorescence intensity increased significantly upon saposin C(0W)binding to synthetic phosphatidylserine [PS(18:1,1)] vesicles at acidicpH. This binding induces change is lipid-concentration dependent andrequires at least one unsaturated fatty acid chain. To evaluate thekinetics of this interaction, stopped-flow experiments are conducted thechange in fluorescence during saposin C/liposome complex formation ismonitored. When saposin C(0W) is mixed with PS(18:1,1) or BPS vesicles,fluorescence of Trp is increased, but the time course of this change isundetectable due to limitation of the machine's capability. Apparently,the interaction of saposin C and unsaturated PS containing membranesoccurs within at least 10 ms.

From CD and emission spectra data, saposin C binds negatively charged,unsaturated phospholipids. This suggests there is an electrostaticinteraction between positively charged residues in saposin C and thenegatively charged membrane surface. This initial interaction isfollowed by the protein embedding into membrane through a hydrophobicinteraction. No shift in emission or change in intensity of Trpfluorescence is observed with the saposin C (0W) and PS(18:0,0) mixture.

Spatial Interactions

To determine the depth of saposin insertion into BPS liposomes,spin-labeled phosphatidylcholines (SLPCs) are incorporated into BPSliposomes with increasing mole percentages (0-50%). SLPCs, hydrophobicfluorescence quenchers, contain doxyl groups which are located atdifferent carbons (n) in the acyl chain: SLPC5 (n=5), SLPC10 (n=10), andSLPC16 (n=16). After addition of Trp-saposins, the protein-liposomemixture (protein:lipid=1:20) is incubated at room temperature for 30minutes, and then, the fluorescence intensity changes are recorded. Forthe Trp-saposins that show the blue-shifts in Table 2, significantquenching effects (30-60%) are observed with BPS/SLPC5 liposomes. Thequenching efficiency is dependent upon the location in the acyl chain ofthe doxyl groups on SLPC. The deeper the doxyl group is in the membrane,the lower quenching efficiency. With BPS/SLPC10, the tryptophanylfluorescence of saposin C (0W) is quenched by 30%.

2. Saposin C-Induced Membrane Fusion

Saposin C is a multifunctional molecule having lysosomal enzymeactivation and neuritogenic activities. Detailed function/structureorganization of saposin C is shown in FIG. 3.

The amino acid residues 51-67 are necessary, but not sufficient, for itsoptimal enzymatic activation function. The disulfide structure andconformational alteration of saposin C upon lipid binding are alsorequired for this activity. Three approaches are used for this study:(1) stopped-flow monitoring reduction of self-quenching resulting fromfusion of fluorescence probe-containing vesicle with non-fluorescentvesicle induced by saposin C; (2) monitoring the lipid vesicle sizechanges upon addition of saposin C to vesicles, the size distribution asdetermined using N4 plus submicron particle sizer (Coulter Co.); (3)monitoring intrinsic fluorescence of Trp-saposin C change duringliposomal fusion. These results define the fusogenic activity regions atthe α-helical domain at amino- and carboxyl-terminus in saposin C, andkinetics of saposin C induced liposomal fusion (see below).

Saposin C Induced Liposomal Fusion

Fluorescence probes have been widely used to determine membrane fusion,such as fluorescence dequenching, and fluorescence resonance energytransfer (FET), and can be used for quantitative and kinetics analyses.The dequenching approach is used to investigate saposin C's fusogenicactivity. Octadecyl rhodamine B (R18) is selected as fluorescence probeand is entrapped in internal aqueous compaitment of liposomal vesiclesby co-sonication with BPS or PS(18:1,1). R18 shows self-quenching athigh concentrations.

Fluorescence increase (dequenching) of R18 occurs upon R18 concentrationdecreases. After non-labeled and labeled vesicles fuse, the R18concentration is diluted, resulting in an increase in intensity offluorescence. R18-labeled vesicles (lipid:R18=96:4, mol:mol) are mixedwith the same lipid vesicles without fluorescence probe. Stopped-flowassays are conducted to quickly mix these vesicles with saposin C orCa²⁺ ion. Time-trace curves are generated for kinetic analysis.Induction of unsaturated PS(18:1,1) membrane fusion by saposin C showsthe same kinetics as those with Ca²⁺. Fusion occurs extensively whenreaction temperature is above the phase Transition temperature (T_(c))of phospholipids. The T_(c) of synthetic PS(18:1) is about −11° C.,while the T_(c) of PS(18:0) is very high (68° C.). Thus, the lipidbilayer phase of PS(18:1,1) is different to that of BPS(18:0 and 18:1)at 24° C. The results indicate that kinetics of saposin C-inducedmembrane fusion is determined by the physical state of the bilayerlipids.

3. Size Change Determination

Electron microscopy (EM) is used for vesicle fusion analysis, since thesize of fused vesicles is bigger than those of non-fused. N4 plussubmicron particle size is used to estimate particle sizes in the rangeof 3 nm to 3 μm since most liposomes fit in this range. Sonicationconditions with a cup sonicator give mono-dispersed BPS-liposomes with˜200 nm in size. Upon addition of saposin C, these vesicles change to alarger size up to 2-3 μm. The size increase is related to vesiclesfusion as shown by the above dequenching experiments. Saposin C enlargesvesicle size at pH 4.7, but not at pH 7.4 over a 10 min period (see FIG.4).

These data suggest a pH-sensitive fusogenic activity of saposin C.Saposin C promotes the size changes at ˜50 nM concentration. To definethe regions responsible for this fusion property, peptides containingonly 50% of the NH₂-terminal or 50% of the COOH-terminal halves insaposin C are tested. Both peptides show fusion activity. These datasuggest linear sequence(s) mediated fusion located on both saposin Cends.

4. Mechanism of Fusion

Protein conformational changes are thought to play a role inprotein-mediated membrane fusion. This fusion mechanism is evaluatedusing saposin C-dependent membrane fusion. First, saposin C-PS(18:1,1)liposome complexes are formed. In this saposin C-anchored membrane,protein conformation is altered. This complex is stable from pH 3 to 10,and in low concentrations of SDS solution. This indicated thatdissociated rate of saposin C from PS vesicles is very slow.

Since the Trp in saposin C(0W) is embedded inside of lipid bilayer, thechange of its signal is indicative of that the surrounding environmentof Trp has been changed. After about 20 to 30 ms, Trp fluorescencesignal decreases to the starting level. This indicates that saposin C inthe complexes interacted with additional PS-vesicles. Shortly afterthis, the signal drops back to starting level signaling on end of thefusion process. These data indicate that saposin C retains the fusogenicactivity even when it bound to lipid membrane. Therefore, aconformational change of saposin C upon lipid binding is not requiredfor its fusogenic activity. This result is consistent with theconclusion that a linear sequence(s) is sufficient to induce membranefusion

XI. Saposin C Gene Optimization and Synthesis

The Saposin C DNA sequence is codon-optimized for expression in E. coli,with consideration of mRNA secondary structure and elimination ofrestriction sites later to be used for subcloning. The restriction sitesNdeI and SalI are added at the 5′ and 3′ ends, respectively, of thegene, and double stop codons are added at the end of the Saposin Ccoding sequence to ensure proper termination of the expressed protein.Gene synthesis is contracted to DNA2.0, and the optimized gene isconfirmed by sequencing and supplied to VTI in the cloning vector “pJ2”.This vector construct is referred to as pJ2-SapCg; the optimized SaposinC gene cassette, bordered by the restriction sites NdeI and SalI, isreferred to as SapCg.

Cloning into pET24a

Cloning of SapCg into pET24a is carried out in the following manner.SapCg is cut out of pJ2-SapCg using restriction sites NdeI and SalI andligated into the expression vector pET24a (Novagen), which has also beencut at those same sites. This ligated construct is transformed into E.coli cloning strain TOP10 (Invitrogen). Selection is carried out withkanamycin (50 mg/L). Colony PCR is carried out to determine whichtransformed colonies carried vector with SapCg insert. One colony ischosen for further work from those that tested positive. Plasmid DNA isprepared by plasmid miniprep kit (Qiagen) from this clone, and thepresence of SapCg insert DNA is confirmed by restriction and sequenceanalysis. This plasmid construct is called pET24a-SapCg.

Shake Flask Induction

The expression construct, pET24a-SapCg, is transformed into competent E.coli expression strain BL21(DE3) (Novagen). Three colonies (clones) arechosen and tested for initial test expression of Saposin C. This smallscale expression is carried out in 125 ml shake-flasks using LB mediawith kanamycin (50 mg/L) for BL21(DE3) clones. Induction is achieved byaddition of IPTG to a final concentration of 1 mM when cell OD600reached approximately 0.6. Samples are taken immediately prior to and atfour hours after induction. Expression of protein of the correct size issimilar for all three BL21 (DE3) clones. Working glycerol seed stocksare prepared for all three clones. One clone is randomly selected forfurther development.

Fermentation Conditions

The clone used for fermentation is pET24a-SapCg (described above)transformed in the E. coli expression strain BL21(DE3). Fermentation isconducted using an 8-liter NBSC BioFlow 3000 fermentor, and consisted offed-batch fermentation with DO-Stat feeding strategy. Initial culturevolume is 5 liters. Batch media and feeding media composition is shownin Tables 1 and 2. The fermentation temperature prior to induction is30° C. Induction is accomplished by addition of IPTG to a finalconcentration of 1 mM at late log phase, accompanied by a temperatureshift to 37° C. The entire fermentation lasted 22 hours, with inductionfor 10 hours.

TABLE 1 Batch media Component Amt/L Unit 1 (NH₄)₂HPO₄ 4 g 2 KH₂PO₄ 13.3g 3 MgSO₄•7H₂O 1.2 g 4 Citric acid 1.7 g 5 Yeast extract 2 g 6 Tracemetal 10 ml solution 7 Glucose•H₂O 22 g 8 Adjust volume to QS ml 5 Lwith Type I water, pH 6.8

TABLE 2 Feeding media Amt/ Component L Unit 1 D-glucose- 660 gmonohydrate 2 Yeast extract 60 g 3 MgSO₄•7H₂O 20 g 4 Type I water QS ml

Inclusion Prep

An inclusion body prep is carried out using paste from the abovefermentation. Approximately 20 g paste is resuspended in a total of 200ml lysis buffer (50 mM Tris pH8, 1 mM EDTA, 100 mM NaCl). Afterresuspension and complete homogenization, microfluidization is used tobreak open cells. The insoluble portion of the cell lysate is pelletedby centrifugation for 60 min at 16,000×g at 4° C. Pellets arehomogenized in a total of 800 ml lysis buffer plus 1% triton X-100 andmixed 45 minutes at room temperature. Centrifugation is carried out for60 min at 16,000×g and 4° C. Two more washes are carried out using lysisbuffer with 1% triton X-100 and one time using lysis buffer withouttriton X-100. Pellets are then resuspended in a total of 600 ml 6M ureapH8.5 (buffered with 20 mM Tris) and stirred at room temperature for 3hrs. Centrifugation is carried out for 60 mM at 16,000×g and 4° C. toclarify sample. The resulting supernatant is used for furtherpurification after confirmation of the presence of Saposin C using aSaposin C specific antibody.

SapC Chromatography and Refolding

The following chromatography, refolding, and concentration steps are allperformed under endotoxin-free conditions. Purification of Saposin Cfrom inclusion bodies is carried out by ion exchange chromatographyusing Q-sepharose Fast Flow resin (GE Amersham). Equilibration buffer(Buffer A) is 6 M urea/0.02M Tris, pH8.5. Elution buffer (Buffer B) is 6M urea/1 M NaCl/0.02 M Tris, pH8.5. Elution is initially carried out bystep gradient, with 5% BufferB/95% BufferA for 10 column volumes, then10% BufferB/90% BufferA for 10 column volumes, followed by a lineargradient from 10% to 100% BufferB over 10 column volumes. All fractionsare collected and retained. Analysis of fractions for presence ofSaposin C is carried out by SDS-PAGE, and the fraction containing themajority of Saposin C is chosen for refolding.

Refolding is carried out by dialysis into McIlvaine buffer (0.05 Mcitric acid/0.1 M phosphate, pH 4.7). Saposin C protein is thenconcentrated to approximately 0.2 mg/ml. This preparation is determinedto be approximately 90% pure by visual examination of SDS-PAGE.

1.-52. (canceled)
 53. A method of delivering an agent to a disease,wherein the method comprises administering to a patient a compositioncomprising: a) a phosphatidylserine, b) a safe and effective amount ofthe agent, and c) a saposin C polypeptide; wherein the disease is abrain cancer or a neuroblastoma.
 54. The method of claim 53 wherein theadministering of the composition comprises a mode of administration,wherein the mode of administration is selected from one or more of thelist consisting of a transdermal patch, enteral delivery, trans-nasaldelivery, intravenous delivery, intramuscular delivery and topicaldelivery.
 55. The method of claim 54 wherein the mode of administrationis intravenous delivery.
 56. The method of claim 53 wherein the molarratio of the saposin C-polypeptide to the phosphatidylserine is betweenabout 1:7 and 1:50.
 57. The method of claim 53 wherein the pH of thecomposition is between about 8 and
 2. 58. The method of claim 53 whereinthe agent is a pharmaceutical agent.
 59. The method of claim 53 whereinthe agent is an imaging agent.
 60. A method for treating a diseasecomprising delivering an agent through a biological membrane, whereinthe method comprises administering to a patient a compositioncomprising: a) a phosphatidylserine, b) a safe and effective amount ofthe agent, and c) a saposin C polypeptide; wherein the disease is abrain cancer or a neuroblastoma.
 61. The method of claim 60 wherein theadministering of the composition comprises a mode of administration,wherein the mode of administration is selected from one or more of thelist consisting of a transdermal patch, enteral delivery, trans-nasaldelivery, intravenous delivery, intramuscular delivery and topicaldelivery.
 62. The method of claim 61 wherein the mode of administrationis intravenous delivery.
 63. The method of claim 60 wherein the molarratio of the saposin C-polypeptide to the phosphatidylserine is betweenabout 1:7 and 1:50.
 64. The method of claim 60 wherein the pH of thecomposition is between about 8 and
 2. 65. The method of claim 60 whereinthe agent is a pharmaceutical agent.
 66. A method for delivering anagent across a membrane of the blood-brain barrier wherein the methodcomprises administration to the membrane a composition comprising: a) aphosphatidylserine, b) a safe and effective amount of the agent, and c)a saposin C polypeptide.
 67. The method of claim 66 wherein theadministering of the composition comprises a mode of administration,wherein the mode of administration is selected from one or more of thelist consisting of a transdermal patch, enteral delivery, trans-nasaldelivery, intravenous delivery, intramuscular delivery and topicaldelivery.
 68. The method of claim 67 wherein the mode of administrationis intravenous delivery.
 69. The method of claim 66 wherein the molarratio of the saposin C-polypeptide to the phosphatidylserine is betweenabout 1:7 and 1:50.
 70. The method of claim 66 wherein the pH of thecomposition is between about 8 and
 2. 71. The method of claim 66 whereinthe agent is a pharmaceutical agent.
 72. The method of claim 66 whereinthe agent is an imaging agent.